Genetic method for controlling sprouting

ABSTRACT

This invention relates to a method of controlling sprout formation in plants and parts thereof including vegetative storage organs. The method involves the use of target and organ specific promoters to control expression of DNA sequences to inhibit sprouting. Sprouting is restored by switching on expression of DNA sequences using inducible promoter regions where sprouting may be controlled by, for example, application of an external chemical stimulus.

The present invention relates to a method of controlling sproutformation in plants and parts thereof including vegetative storageorgans.

Potato tubers are of major economic importance. They represent acarbohydrate resource for many diets and are used as a basis for avariety of processed products. Besides starch, tubers containhigh-quality proteins, substantial amounts of vitamins, minerals andtrace elements. Continuous production of potato tubers throughout theyear is impossible in most regions where potatoes are grown. As aconsequence storage of the harvested tubers is required.

One of the potentially most damaging phenomena during storage ispremature sprouting. Long term storage involves cooling, forcedventilation and use of chemical sprouting suppressants. The problemsdirectly linked to long term storage are manifold.

Cooling, usually done in Northern Europe by ventilation with air atambient temperature is one of the methods to inhibit sprouting. Apartfrom the associated costs, longer term cooling at 4° C. gives rise tothe problems of cold sweetening and melanisation (darkening).

Chemical sprouting suppressants are currently the only possibility forinhibiting sprouting in potatoes destined for processing and freshconsumption, since low temperature storage leads to unacceptableaccumulation of reducing sugars. However, in recent years, questionshave arisen as to the environmental and nutritional impact of chemicalsuppressants such as chlorinated hydrocarbons. There is therefore a realneed for an alternative method of controlling sprouting in vegetativestorage organs such as tubers.

An alternative approach to delay sprouting would be the use oftransgenic plants with a prolonged quiescence period. Sprouting ofpotato tubers involves several independent steps which might be targetsfor genetic engineering. The first step is the mobilisation of reserves,mainly starch. Starch breakdown occurs in amyloplasts and is mediated bystarch phosphorylase and/or amylases. In the next step following starchbreakdown, the resultant hexoses and/or hexose-phosphates have to beexported from amyloplasts. After transfer into the cytosol the producedhexoses and hexose-phosphates are distributed between glycolysis andsucrose synthesis. The third step is the formation of sucrose in thecytosol. Sucrose synthesis is energy dependent thus glycolysis andrespiration are required. The fourth step is the transport of sucrose tothe developing sprout. Finally the imported sucrose is utilised in thesprout to support growth and development.

We have now developed a means of controlling sprouting in vegetativestorage organs such that sprouting may be turned off and on without anyundesirable side effects such as yield loss. This new method involvesthe targeted expression of genes resulting in the disruption ofsprouting in combination with gene switch technology to restoresprouting when required.

According to a first aspect of the present invention there is provided amethod for the selective induction or suppression of sprouting in aplant comprising incorporating, preferably stably incorporating, intothe genome of said plant by transformation a DNA construct comprising afirst polynucleotide sequence comprising at least one DNA sequenceoperably linked to a tissue or organ selective promoter region andoptionally to a transcription terminator region and a secondpolynucleotide sequence comprising at least one DNA sequence operablylinked to and controlled by a controllable promoter region andoptionally to a transcription terminator region whereby the DNAsequence(s) in said first polynucleotide sequence is expressed duringdormancy of the vegetative organ derived from said transgenic plantresulting in effective suppression of sprouting and the said suppressionis neutralised by inducing expression of the DNA sequence(s) in saidsecond polynucleotide sequence from said controllable promoter region byexternal application of an inducing substance such that restoration ofsprouting of said vegetative storage organ is dependent on theapplication of the inducer.

As used herein the term “tissue or organ selective promoter region”denotes those promoter regions which yield preferential expression ofthe DNA sequence(s) of interest in the desired tissue or organs.

The DNA sequences in the DNA construct may be endogenous or heterologouswith respect to the transformed host.

Examples of DNA sequences which may be used in the method of the presentinvention to control sprouting include those DNA sequences coding forproteins involved in the mobilisation of reserves during dormancy suchas the breakdown of storage compounds e.g starch breakdown, i.e starchphosphorylase, amylase (e.g. α or β amylase) and maltase; e.g inglycolysis and subsequent metabolism e.g phosphofructokinase,hexokinase; in sucrose biosynthesis e.g sucrose synthase; in thetransport of reserves during dormancy such as in phloem loading e.gATPase; in long distance phloem transport and in phloem unloading e.ginorganic pyrophosphorylase (iPPase); and in the utilisation of reservesduring dormancy such as in assimilate breakdown e.g the breakdown ofsucrose in the growing sprout, i.e invertase; and in the utilisation ofassimilates e.g utilisation of sucrose-derived metabolites, in theprovision of energy required for sprout formation e.g. DNA sequencescoding for proteins involved in mitochondrial function such as inrespiration, such as mitochondrial enzymes and transport proteins suchas translocators e.g. adenine nucleotide translocator (ANT) and malateoxoglutarate translocator (MOT) and inhibitors thereof such asuncoupling proteins. Examples of useful DNA sequences also include anyother sequences which are involved in potato sprouting

Examples of preferred DNA sequences which may be used in the method ofthe present invention to control sprouting include those resulting inthe production of sense, anti-sense or partial sense sequence(s) to,and/or coding for, proteins involved in the mobilisation and/orutilisation of sucrose, in potato sprouting and in mitochondrialfunction such as in respiration.

Examples of particularly preferred DNA sequences include those codingfor an invertase derived from plant, bacterial or fungal sources e.g.from yeast, a pyrophosphatase derived from plant, bacterial or fungalsources and proteins involved in mitochondrial function such as MOT andANT derived from plant, bacterial or fungal sources which are describedhereinafter.

Suppression of sprouting may be achieved in a variety of ways. The firstDNA sequence(s) may be expressed during dormancy of the vegetativestorage organ and then down-regulated when sprouting is desired. Whensprouting is desired expression of the second DNA sequence(s) is turnedon leading to down regulation of the first DNA sequence and consequentlyrestoration of sprouting.

Down regulation of a desired DNA sequence(s) may be achieved usingmethods well known in the art such as, for example, by use of repressorproteins, sense, anti-sense, partial-sense, and expression of acomplementary protein. Examples of suitable operator/repressor systemsinclude for example the lac, tet or lambda 434 systems and mutantsthereof such as the Lac IΔ His mutant (Lehming, N., Sartoris, J.,Niemoeller, M., Genenger, G., v. Wilcken-Bergman, B. and Muller-Hill,Benno (1987), EMBO J. 6(10) 3145-3153—where the mutant has a change inamino acid 17 of Lac I altering tyrosine for histidine). Alternatively,an Amplicon™ may be used to down-regulate genes (Angell, S. M.,Baulcombe, D. C., (1997) 16, 3675-3684). In this regard, the cDNA ofreplicating potato virus (PVX) RNA which has a transgene insertedtherein is used whereby transiently expressed RNA sharing homology withthe transgene is suppressed.

Alternatively, expression of the DNA sequence(s) in the firstpolynucleotide sequence may result in the production of a sense,anti-sense or partial-sense sequence(s) which acts to suppress a geneinvolved in sprouting or in the expression of an Amplicon™. In this casesprouting is restored by switching on expression of the DNA sequence(s)in the second polynucleotide sequence which results in production of theprotein or a corresponding protein to that, the production of which wassuppressed by the sense, anti-sense or partial-sense sequence(s) in thefirst DNA sequence. Sprouting may also be restored by means of asuitable operator/repressor system.

Where either or both of the polynucleotide sequences in the constructcomprise more than one DNA sequence it is preferable that they are notidentical to avoid any co-suppression effects.

Expression of the DNA sequence(s) in the first polynucleotide sequenceis under the control of a tissue or organ selective promoter to ensuretargeted expression of the DNA sequence whereby expression is induced inan organ or tissue specific manner. Examples of tissue selectivepromoters include phloem selective promoters e.g. the rolC promoter, andexamples of organ selective promoters include tuber specific promoters,such as the patatin promoter. The use of tissue or organ selectivepromoters such as the rolC and tuber promoters is particularlypreferred.

The DNA sequence(s) in the second polynucleotide sequence of theconstruct is under the control of a controllable promoter region.

As used herein the term “controllable promoter region” includespromoters which may be induced chemically. The use of a promotersequence which is controlled by the application of an external chemicalstimulus is most especially preferred. The external chemical stimulus ispreferably an agriculturally acceptable chemical, the use of which iscompatible with agricultural practice and is not detrimental to plantsor mammals.

The controllable promoter region most preferably comprises an inducibleswitch promoter system as such as, for example, a two component systemsuch as the alcA/alcR gene switch promoter system described in ourpublished International Patent Application No. WO 93/21334; the GSTpromoter as described in our published International Patent ApplicationNos. WO 90/08826 and WO 93/031294; and the ecdysone switch system asdescribed in our published International Patent Application No. WO96/37609, the teachings of which are incorporated herein by reference.Such promoter systems are herein referred to “switch promoters”. Theswitch chemicals used in conjunction with the switch promoters areagriculturally acceptable chemicals making this system particularlyuseful in the method of the present invention. In the case of thealcA/alcR promoter switch system the preferred chemical inducer isethanol in either liquid or more preferably in the vapour form. One ofthe main advantages of the use of ethanol vapour is that only smallquantities of ethanol are required and that high levels of expressionare achieved. Full details of switch chemicals are provided in thepatent applications listed immediately above.

Suitable transcription terminators which may be used are also well knownin the art and include for example the nopaline synthase terminator andoctopine synthase terminators. The promoter is most desirably a latetuber specific promoter which is active late in the dormancy period i.ejust before sprouting.

The controllable promoter region for use in the method of the presentinvention is preferably the GST or alcA/alcR promoter switch system.Restoration of sprouting is preferably achieved using switchableantisense or switchable sense or partial sense methods as is describedmore fully herein or alternatively by use of an Amplicon™ or by means ofa suitable operator/repressor system. Down-regulation of gene activitydue to partial sense co-suppression is described in our InternationalPatent Application No. WO 91/08299 the teachings of which areincorporated herein and this may be avoided if necessary by using genesequences derived from different organisms.

According to a second aspect of the present invention there is provideda DNA construct comprising a first polynucleotide sequence comprising atleast one DNA sequence operably linked to a tissue or organ selectivepromoter region and optionally to a transcription terminator region anda second polynucleotide sequence comprising at least one DNA sequenceoperably linked to and controlled by a controllable promoter region anda transcription terminator region wherein said first polynucleotidesequence comprises a DNA sequence coding for a protein involved inmobilisation and/or utilisation of sucrose and said secondpolynucleotide sequence comprises a DNA sequence which is a sense, ananti-sense or partial sense sequence corresponding to said protein or aDNA sequence which is capable of causing suppression of said protein.

According to a third aspect of the present invention there is provided aDNA construct comprising a first polynucleotide sequence comprising atleast one DNA sequence operably linked to a tissue or organ selectivepromoter region and optionally to a transcription terminator region anda second polynucleotide sequence comprising at least one DNA sequenceoperably linked to and controlled by a controllable promoter region anda transcription terminator region wherein said first polynucleotidesequence comprises a first DNA sequence coding for a protein involved inmobilisation and/or utilisation of sucrose and a further DNA sequencecoding for an operator sequence operably linked to the first DNAsequence and the second polynucleotide sequence comprises a DNA sequencecoding for a repressor protein capable of binding to said operatorsequence.

According to a fourth aspect of the present invention there is provideda DNA construct comprising a first polynucleotide sequence comprising atleast one DNA sequence operably linked to a tissue or organ selectivepromoter region and optionally to a transcription terminator region anda second polynucleotide sequence comprising at least one DNA sequenceoperably linked to and controlled by a controllable promoter region anda transcription terminator region wherein said first polynucleotidecomprises a DNA sequence(s) which is a sense, anti-sense or partialsense sequence corresponding to a protein involved in potato sproutingor a DNA sequence which is capable of causing suppression of a proteininvolved in potato sprouting and said second polynucleotide sequencecomprises a DNA sequence(s) coding for a protein involved in potatosprouting.

According to a fifth aspect of the present invention there is provided aDNA construct comprising a first polynucleotide sequence comprising atleast one DNA sequence operably linked to a tissue or organ selectivepromoter region and optionally to a transcription terminator region anda second polynucleotide sequence comprising at least one DNA sequenceoperably linked to and controlled by a controllable promoter region anda transcription terminator region wherein said first polynucleotidecomprises a first DNA sequence(s) which is a sense, anti-sense orpartial sense sequence corresponding to a protein involved in potatosprouting or a DNA sequence which is capable of causing suppression of aprotein involved in potato sprouting and a further DNA sequence codingfor an operator sequence operably linked to the first DNA sequence andsaid second polynucleotide sequence comprises a DNA sequence(s) codingfor a repressor protein capable of binding to said operator sequence.

According to a sixth aspect of the present invention there is provided aDNA construct comprising a first polynucleotide sequence comprising atleast one DNA sequence operably linked to a tissue or organ selectivepromoter region and optionally to a transcription terminator region anda second polynucleotide sequence comprising at least one DNA sequenceoperably linked to and controlled by a controllable promoter region anda transcription terminator region wherein said first polynucleotidecomprises a DNA sequence(s) which is a sense, anti-sense or partialsense sequence corresponding to a protein involved in mitochondrialfunction or a DNA sequence which is capable of causing suppression of aprotein involved in mitochondrial function and said secondpolynucleotide sequence comprises a DNA sequence(s) coding for a proteininvolved in mitochondrial function.

According to a seventh aspect of the present invention there is provideda DNA construct comprising a first polynucleotide sequence comprising atleast one DNA sequence operably linked to a tissue or organ selectivepromoter region and optionally to a transcription terminator region anda second polynucleotide sequence comprising at least one DNA sequenceoperably linked to and controlled by a controllable promoter region anda transcription terminator region wherein said first polynucleotidecomprises a first DNA sequence(s) which is a sense, anti-sense orpartial sense sequence corresponding to a protein involved inmitochondrial function or a DNA sequence which is capable of causingsuppression of a protein involved in mitochondrial function and afurther DNA sequence coding for an operator sequence operably linked tothe first DNA sequence and said second polynucleotide sequence comprisesa DNA sequence(s) coding for a repressor protein capable of binding tosaid operator sequence.

We have found the following combination of DNA sequences to beparticularly suitable for use in the method of the invention: by placinga DNA sequence coding for an invertase under the control of a phloemselective promoter such as the rolC promoter, it is possible to targetexpression of the DNA sequence to the phloem and effectively represssprouting and to then restore sprouting by switching on a DNA sequencecoding for invertase anti-sense using the alcA/alcR chemical switchpromoter. Sucrose concentration in the phloem from the leaf is so highthat the effects of invertase expression are effectively swampedavoiding any unwanted side effects. This contrasts with the situation inthe sprout phloem where expression of invertase has a dominant effectwith the result that sucrose is broken down and sprouting is effectivelyinhibited.

A further useful combination is a DNA sequence coding for an inorganicpyrophosphatase (iPPase) under the control of a tuber promoter. Uptakeof sucrose and transport in the phloem is an energy requiring processand by inhibiting the provision of energy by expressing the DNA sequencecoding for inorganic pyrophosphatase it is possible to inhibit theuptake process. The inhibition can be reversed by using, for example, analcA/alcR chemically induced switch promoter to switch on a DNA sequencecoding for an antisense, sense or partial sense sequence to iPPase andsprouting is restored. Again the use of a tissue or organ selectivepromoter ensures that the inhibition of sucrose uptake and transport inthe phloem does not occur in the whole plant but only in the tuberthereby minimising any deleterious effects in the plant.

In both cases, an alternative means of restoring sprouting is by the useof an Amplicon™ where transiently expressed RNA sharing homology withthe transgene is suppressed. Such a transgene could, for example, be acDNA for an invertase or iPPase. A further alternative means ofrestoring sprouting is by the use of a suitable operator/repressorsystem.

We have also found that by selectively inhibiting the provision ofenergy required for sprout growth and development in the tuber byplacing a DNA sequence coding for sense, antisense or partial sense to aDNA sequence coding for a protein involved in mitochondrial function,such as the adenosine nucleotide translocator protein (ANT) ormitochondrial oxoglutarate translocator (MOT), under the control of atuber selective promoter sprouting may be inhibited without unwantedside effects. Alternatively, a DNA sequence which causes suppression ofsuch proteins may be used. One way in which reversal of the inhibitionmay be achieved is by switching on expression of a second DNA sequencethe product of which is complementary to the first DNA sequence, forexample a DNA sequence coding for ANT derived from a different sourcepreferably from Arabidopsis may be used to counteract the effect of theANT antisense expression. In the case of MOT a suitable complementarysequence may be derived from Panicum miliaceum as is described byTaniguchi and Sugiyama in Plant Molec. Biol. 30, 51-64 (1996).Alternatively, a suitable operator/repressor system may be used toreverse inhibition. As above the alcA/alcR chemical switch promoter maybe used. The above examples are described more fully herein.

According to some embodiments of the present invention the firstpolynucleotide sequence comprises a further DNA sequence coding for anoperator sequence operably linked to the first DNA sequence and thesecond polynucleotide sequence comprises a DNA sequence coding for arepressor capable of binding to the operator sequence under the controlof a switch promoter such that application of the inducer results inexpression of the DNA sequence coding for the repressor whichsubsequently binds to the operator and expression of the first DNAsequence in the first polynucleotide sequence is switched off. Anexample of such a system is the lactose operator and repressor proteinas is described in published International patent Application No. WO90/08830. Other examples include the tetracycline and lambda 434operator/repressor systems.

Plant cells may be transformed with recombinant DNA constructs accordingto a variety of known methods for example, Agrobacterium Ti plasmids,electroporation, microinjection and by microprojectile gun. Thetransformed cells may then, in suitable cases, be regenerated into wholeplants in which the new nuclear material is incorporated, preferablystably incorporated, into the genome. Both transformed monocotyledonousand dicotyledenous plants may be obtained in this way.

According to an eighth aspect of the present invention, there isprovided a plant cell transformed with any one of the DNA constructsdefined above.

According to a nineth aspect of the present invention there is alsoprovided a whole plant transformed with a DNA construct according to theabove aspects of the present invention wherein said DNA construct isincorporated, preferably stably incorporated, into the genome of saidplant.

The invention still further includes, according to a tenth aspect of thepresent invention, progeny of the plants of the preceding paragraphwhich progeny comprise a DNA construct according to the above aspects ofthe present invention incorporated, preferably stably incorporated, intotheir genome and the seeds and tubers of such plants and such progeny.

The method of the present invention is particularly suitable forcontrolling sprouting in potato tubers.

In a preferred embodiment the invention provides a method for theselective induction or suppression of sprouting in potatoes comprisingstably incorporating into the genome of said potato by transformation aDNA construct comprising a first polynucleotide sequence comprising atleast one DNA sequence operably linked to a tissue or organ selectivepromoter region and optionally to a transcription terminator region anda second polynucleotide sequence comprising at least one DNA sequenceoperably linked to and controlled by a controllable promoter region andoptionally to a transcription terminator region whereby the DNAsequence(s) in said first polynucleotide sequence is expressed duringdormancy of the tuber derived from said transgenic potato resulting ineffective suppression of sprouting and the said suppression isneutralised by inducing expression of the DNA sequence(s) in said secondpolynucleotide sequence from said controllable promoter region byexternal application of an inducing substance such that restoration ofsprouting of said tuber is dependent on the application of the inducer.

We have also identified five particularly preferred DNA sequences whichwe believe may also be especially useful in the method of the presentinvention. We have identified these DNA sequences as being inducedduring tuber storage and we have designated these as 16-3 (sequence 2),16-8 (sequence 3), 10-1 (sequence 4) and AC4 (sequence 5), M-1-1 (MOT)(sequence in FIG. 19) and a MOT variant (sequence 6—having an EMBLAccession number X99853). The DNA sequences and their isolation aredescribed fully in the accompanying examples. The present inventiontherefore provides, according to a further aspect, the use of all orpart of the DNA sequences from clones 16-3, 10-1, AC4, 16-8, M-1-1 andthe MOT variant in a method according to the invention to controlsprouting in plants.

The DNA sequences of 16-3, 16-8, AC4 and M-1-1 are believed to be newand a twelfth aspect of the present invention extends to polynucleotidescomprising nucleotides 1 to 870 in sequence 2 (corresponding to 16-3),nucleotides 1 to 712 in sequence 3 (corresponding to 16-8) ornucleotides 1 to 386 in sequence 5 (corresponding to AC4) or nucleotides1 to 1351 in sequence FIG. 19 (corresponding to M-1-1 encoding a MOT)and further to the protein products encoded thereby and to thoseproteins having a substantially similar activity and having an aminoacid sequence which is at least 85% similar to the said product. It ispreferred that the degree of similarity is at least 90% and it is morepreferred that the degree of similarity is 95% and it is most preferredthat the degree of similarity is 97%.

A particularly preferred embodiment of the polynucleotides consists ofnucleotides 55 to 751 in sequence 2, nucleotides 87 to 473 in sequence3, and to nucleotides 192 to 164 in FIG. 19 and further to thetranslation products encoded thereby and to those proteins having asubstantially similar activity and having an amino acid sequence whichis at least 85% similar to the said product. It is preferred that thedegree of similarity is at least 90% and it is more preferred that thedegree of similarity is 95% and it is most preferred that the degree ofsimilarity is 97%.

As used herein the term “degree of similarity” is used to denotesequences which when aligned have similar (identical or conservativelyreplaced) amino acids in like positions or regions, where identical orconservatively replaced amino acids are those which do not alter theactivity or function of the protein as compared to the starting protein.For example, two amino acid sequences with at least 85% similarity toeach other have at least 85% similar (identical or conservativelyreplaced amino residues) in a like position when aligned optimallyallowing for up to 3 gaps, with the proviso that in respect of the gapsa total of not more than 15 amino acid resides is affected. The degreeof similarity may be determined using methods well known in the art(see, for example, Wilbur, W. J. and Lipman, D. J. “Rapid SimilaritySearches of Nucleic Acid and Protein Data Banks.” Proceedings of theNational Academy of Sciences USA 80, 726-730 (1983) and Myers E. andMiller W. “Optimal Alignments in Linear Space”. Comput. Appl. Biosci.4:11-17(1988)). One programme which may be used in determining thedegree of similarity is the MegAlign Lipman-Pearson one pair method(using default parameters) which can be obtained from DNAstar Inc, 1228,Selfpark Street, Madison, Wis., 53715, USA as part of the Lasergenesystem.

According to a thirteenth aspect of the present invention there isprovided polynucleotide sequence(s) encoding a protein having asubstantially similar activity to that encoded by nucleotides providedin sequences 2, 3 and 5 and FIG. 19, which polynucleotide iscomplementary to one which still hybridises with the sequence comprisedby that provided in sequences 2, 3 or 5 or FIG. 19 when incubated at orbetween low and high stringency conditions. In general terms, lowstringency conditions can be defined as 3×SSC at about ambienttemperature to about 65 C. and high stringency conditions as 0.1×SSC atabout 65° C. SSC refers to the buffer 0.15M NaCl, 0.015M trisodiumcitrate and 3×SSC is three times as strong as SSC and 0.1×SSC is onetenth of the strength of SSC.

The invention further provides polynucleotide sequence(s) encoding aprotein having a substantially similar activity to that encoded bynucleotides 55 to 751 in sequence 2, nucleotides 87 to 473 in sequence3, or to nucleotides 192 to 1164 in FIG. 19, which polynucleotide iscomplementary to one which still hybridises with the sequence comprisedby nucleotides 55 to 751 in sequence 2, nucleotides 87 to 473 insequence 3, or to nucleotides 192 to 1164 in FIG. 19 when incubated ator between low and high stringency conditions. In general terms, lowstringency conditions can be defined as 3×SSC at about ambienttemperature to about 65 C. and high stringency conditions as 0.1×SSC atabout 65° C. SSC refers to the buffer 0.15M Na Cl, 0.015M trisodiumcitrate and 3×SSC is three times as strong as SSC and 0.1×SSC is onetenth of the strength of SSC.

The polynucleotides according to the present invention depicted insequences 2, 3 and 5 and FIG. 19 may be operably linked to a promoterregion which may be homologous or heterologous to the polynucleotide andthe present invention extends to such constructs. The present inventionalso extends to a DNA construct comprising said polynucleotides furthercomprising a region encoding a peptide which is capable of targeting thetranslation products of the polynucleotide to desired cellular orsub-cellular locations. The invention further provides a vectorcomprising said polynucleotide sequence as described in sequences 2, 3,5 and FIG. 19 preferably operably linked to a promoter region andoptionally to a transcription terminator and or a targeting sequence asdescribed above.

The sequences provided herein for 16-3 (SEQ ID NO. 2), 16-8 (SEQ ID NO.3), 10-1 (SEQ ID NO. 4), AC-4 (SEQ ID NO. 5), M1-1 (SEQ ID NO. 27) andthe MOT variant are cDNA sequences and may, according to a furtheraspect of the present invention, be used as probes for the isolation andidentification from genomic libraries of sequences upstream of the 5′region which contain the natural promoter region. The promoter regionmay then be identified, isolated and sequenced.

According to a fifteenth aspect of the present invention there isprovided a host cell transformed with a DNA construct comprising apolynucleotide sequence as described in sequences 2, 3 and 5 and FIG. 19or a vector described above comprising said polynucleotide sequence. Thehost cell is preferably a plant cell as described previously and thepresent invention extends also to whole plants having incorporated,preferably stably incorporated, into their genome a polynucleotidesequence, DNA construct or vector as described above, and to seeds,tubers and progeny of said plants.

According to a sixteenth aspect of the present invention there isprovided a DNA construct comprising a polynucleotide sequence comprisinga switch promoter system operably linked to a polynucleotide sequencecomprising a sense, antisense or partial sense transcription constructwherein when expression of said polynucleotide sequence is switched onfrom the switch promoter the resulting expression of said sense,antisense or partial sense sequence leads to down regulation of theexpression of a further polynucleotide sequence encoding a transgene.

In a seventeenth aspect the present invention provides a method ofcontrolling the expression of a transgene comprising transforming a hostcell with a DNA construct comprising a switch promoter system operablylinked to a polynucleotide sequence comprising a sense, antisense orpartial sense transcription construct, and a further DNA constructcomprising a coding sequence coding for a transgene and controllingexpression of the polynucleotide sequence from said switch promoter suchthat the resulting expression of the said sense, antisense or partialsense construct leads to down regulation of the expression of saidtransgene.

As used herein the term “transgene” is used to denote a gene which isforeign or heterologous to the transformed host cell.

In a preferred embodiment of the above aspects the present inventionprovides a DNA construct comprising the alcA/alcR switch promoteroperably linked to a polynucleotide sequence comprising a sense,antisense or partial sense transcription construct.

The present invention also extends to a vector comprising said DNAconstructs according to the above aspects of the invention.

According to an eighteenth aspect of the present invention there isprovided a host cell transformed with a DNA construct comprising apolynucleotide sequence comprising a switch promoter which may beswitched on by the application of a chemical stimulus operably linked toa polynucleotide sequence comprising a sense, antisense or partial sensetranscription construct wherein when expression of said polynucleotidesequence is switched on from the switch promoter the resultingexpression of said sense, antisense or partial sense sequence leads todown regulation of the expression of a further polynucleotide sequenceencoding a transgene.

The host cell is preferably a plant cell as described previously and thepresent invention extends also to whole plants derived therefrom havingincorporated, preferably stably incorporated, into their genome apolynucleotide sequence, DNA construct or vector as described above, andto seeds, tubers and progeny of said plants.

The use of switch promoter systems to control expression of the sense,antisense or partial sense construct has many applications.Down-regulation of a gene, the expression of which gives rise to alethal or inhibitory effect may be controlled using switchable sense,antisense or partial sense to facilitate the identification of suitableherbicide targets. Switchable down regulation using sense, antisense orpartial sense sequences may also be used to identify mechanisms of cellablation.

The present invention therefore provides according to a nineteenthaspect a method of identifying a site which may be a suitable target forinteraction with a herbicide comprising the steps of transforming aplant with a polynucleotide sequence comprising a first DNA sequencewhich is capable of affecting the expression of DNA at said target sitewherein expression of said first DNA sequence is under the control of aswitch promoter; controlling expression of said DNA sequence from saidswitch promoter such that the expression of the DNA coding for theherbicide target site is down regulated and determining the effects ofsaid down regulation on the plant viability.

The types of effects which would be monitored include the time periodfor which down regulation at the target site must be maintained and whatlevel of down regulation is required and on the basis of the resultsobtained it can be decided whether the target site would be suitable asa target site for a herbicide.

We have most unexpectedly found that the STLS-1 leaf promoter sequenceacts as an enhancer of gene expression in tubers and the use of theSTLS-1 sequence as an enhancer of gene expression in tubers forms afurther aspect of the present invention.

In a twentieth aspect the present invention therefore provides a methodof enhancing gene expression in tubers comprising transforming a tuberplant cell with a polynucleotide sequence comprising a DNA sequencecoding for all or part of the STLS-1 leaf promoter operably linked to afurther promoter region.

The STLS-1 leaf promoter is known in the art (Eckes et al (1986) Mol.Gen. Genet. 205, 14-22) and is described in the accompanying examples.All or part of the DNA sequence coding for the STLS-1 leaf promoter maybe used as an enhancer according to the invention. Active fragments ofSTLS-1 may be identified using techniques well known in the art such asrestriction enzyme digestion followed by analysis of enhancement of geneexpression of the fragments thus obtained. The STLS-1 promoter sequencemay be inserted either upstream i.e. at the 5′ end or downstream i.e. atthe 3′ end of the further promoter region. Insertion of the STLS-1sequence upstream of the promoter region is especially preferred. In aparticularly preferred embodiment of this aspect of the invention theSTLS-1 sequence is inserted upstream of the 35S CaMV promoter.

In a twenty-first aspect the present invention provides tubers, whichare preferably potato tubers, derived from transgenic plants which donot sprout unless treated with a chemical inducer.

According to a twenty-second aspect of the present invention, there isprovided a polynucleotide sequence comprising all or part of at leastone of the sequences depicted in FIG. 25, 26 or 27 and polynucleotideshaving the same function as the sequence which is depicted in FIG. 25,26 or 27 which polynucleotide is complementary to one which stillhybridises with the sequence comprised by that provided in FIG. 25, 26or 27 when incubated at or between low and high stringency conditions.Such sequences are preferably tuber specific promoters.

According to a twenty-third aspect of the present invention, there isprovided a method of controlling gene expression of a plant or a partthereof comprising transforming a plant cell with a chemically inducibleplant gene expression cassette comprising a first promoter operativelylinked to a regulator sequence derived from the alc R gene and acontrollable promoter derived from the alc A gene promoter operativelylinked to a target gene, wherein the controllable promoter is activatedby the regulator protein in the presence of alcohol vapour therebycausing expression of the target gene.

The present invention will now be described by way of the followingnon-limiting examples and with reference to the accompanying figures inwhich:

FIG. 1: (SEQ ID NOS.: 9, 20, 21, and 22, respectively) shows a diagramof the construction of plasmid pBIN-IN8.

FIG. 2: shows a schematic drawing of plasmid PPA-2.

FIG. 3: shows a photograph of wild type (Desiree) and transgenic potatoplants containing the phloem specific cytosolic invertase (genotypeDIN-87, DIN-90 and DIN-30) following prolonged storage in the dark atroom temperature.

FIG. 4: shows western blot analysis of protein extracts from potatotubers of control plants and PPaII-2, -3, and -5 and PPaI-2 and PPaI-55with an antibody raised to inorganic pyrophosphatase. Lanes 1 and 2 aresamples from two independent tubers.

FIG. 5: shows photographs of tubers harvested from wild type andtransgenic plants after storage for five months at room temperature andin the dark. A: wild type control (Desiree); B: transgenic plantPPaII-2; C: transgenic plant PPaII-3; D: transgenic plant PPaII-5.

FIG. 6: shows: A: diagram of plasmid pJIT 166 B: diagram of pAGS/pUC GUSreporter gene construct

FIG. 7: shows a map of plasmid AlcR/AGUS.

FIG. 8: Tissue culture grown potato plants were transferred into thegreenhouse following cultivation for 8 weeks in 2.51 pots. Alcexpression was induced via watering the plants three times (day 0, 1 &2) with 50 ml of a 5% ethanol solution. On day 4 following the initialinduction stolons and developing tubers were harvested and GUS activitywas visualized using the histochemical staining procedure. 0 day, priorinduction; 4 days, 4 days after initial induction shows histochemicaldetection of alc:GUS activity in stem, roots and stolons.

1: non-induced stolon, 2: swelling tuber, 3: developing tuber and 4:mature tuber.

FIG. 9: shows a photograph of potato tubers after histochemicaldetection of alc:GUS activity following ethanol vapour treatment.

A: 0 days, B: 3 days, C: 7 days, D, untreated control, E, 7 days aftertreatment.

FIG. 10: shows a map of plasmid pGSTTAK.

FIG. 11: shows a histogram analysis of GUS activity in fully developedleaves of GST-GUS transformed plants after cultivation for 14 days onMS-medium containing 0% ▪ 0.4 2.0% □ and 10% R-25788.

FIG. 12: (SEQ ID NOS.: 23 and 24) shows a diagram of the construction ofplasmid SQ03.

FIG. 13: shows a diagram of the construction of plasmid SQ-01.

FIG. 14: shows a diagram of the construction of plasmid SQ-02.

FIG. 15: (SEQ ID NOS.: 13 and 14) shows a diagram of the cloning ofpotato ANT.

FIG. 16: shows accumulation of UBL-,GTP-binding-,AC4-, and 16-8-specifictranscripts in different areas of sprouting tubers.

FIG. 17: shows accumulation of UBL-,GTP-binding-, 16-8-, andMOT-specific transcripts in different areas of sprouting tubers.

FIG. 18: (SEQ ID NOS.: 25 and 26) shows a diagram of the construction ofan antisense MOT construct.

FIG. 19: (SEQ ID NOS.: 27 and 33) shows the DNA sequence encoding MOTisolated from potato.

FIG. 20: (SEQ ID NOS.: 34 and 35) shows sequence homology between theprotein encoded by clone M-1-1 (MOT) and Panicum miliaceum mitochondrialoxoglutarate.

FIG. 21: shows the strategy for cloning the lac operator sequence into aRolC-invertase plasmid.

FIG. 22: shows the strategy for cloning Lac I into an Alc switch binaryvector and ligation to RolCopINV.

FIG. 23: (SEQ ID NO.: 36) shows the isolation of the UBL-1 promoter byPCR.

FIG. 24: shows the isolation of the MOT-promoters by PCR.

FIG. 25: (SEQ ID NO.: 37) shows the UBL-1 promoter nucleic acidsequence.

FIG. 26: (SEQ ID NO.: 38) shows the MOT3 promoter nucleic acid sequence.

FIG. 27: (SEQ ID NO.: 39) shows the MOT6 promoter nucleic acid sequence.

FIG. 28: shows a CAT assay of ALC-CAT tobacco leaves from plantsenclosed with an ethanol source for 24 hours. The numbers above thecolumns represent ng ethanol/ml headspace.

FIG. 29: shows the kinetics of CUS RNA transcript in 35S-Alc-GUS potatotubers after ethanol induction. The outer part refers to the part whichis 1-3 mm beneath the skin of the potato tuber, the remaining part ofthe potato being referred to as the inner part. The induction wasperformed in 40 liters of plastic chamber tightly sealed with rubber for1 week. The ethanol concentration was 0.02% gas phase (8 ml of 96%ethanol/401) and 20 μg of the total RNA was loaded onto each slot.

FIG. 30 shows the kinetics of GUS transcript and activity in 35S-Alc-GUSpotato tubers induced by 1% ethanol.

EXAMPLES I. Exemplification of Sprout Inhibition 1. Inhibition of PotatoTuber Sprouting via the Expression of Phloem-specific Invertase

1.1. Construction of Plasmid pBIN-RolC

The rolC promoter from Agrobacterium rhizogenes was cloned by polymerasechain reaction (PCR) following the instructions of the manufacturer(Perkin Elmer, Ueberlingen, Germany). The temperature profile of the PCRcycle (40 cycles) was as follows: 1 min at 95° C., 1 min at 45° C. and 2min at 72° C. Plasmid DNA containing the rolC promoter was isolated fromA. rhizogenes bearing the plasmid pABC002 (Schmülling et al., Plant Cell1, 665-670 (1989)) using standard procedures (Sambrook et al., A CloningManual Cold Spring Harbor Laboratory Press 1989). Syntheticoligonucleotides were synthesised based on the published sequence of therolC promoter fragment (Slightom et al., J. Biol Chem 261 (1) 108-121,1986). The sequences (SEQ ID NOS.: 7 and 8) of the primers were: 5′-rolCprimer D(GGAATTCGATACGAAAAAGGCAAGTGCC AGGGCC) and 3′-rolC primerd(CCCATG GTACCCCATAACTCGAA GCATCC). The amplified DNA was cloned intothe PCR vector pCR1000™ (Invitrogen, Norwalk, Conn.). To excludemutations of the amplified DNA during the PCR cycles, the clone wassequenced using the dideoxy method. The 1150-bp promoter fragment wassubsequently cloned into a plant expression cassette pBINAR (Höfgen andWillmitzer Plant Sci. 66, 221-230 1990) by replacement of the 35SCauliflower mosaic virus promoter sequence (Franck et al., Cell 21285-294 (1980)) through the rolC promoter using 5′-restriction site ofEcoRI and the 3′-restriction site of Asp718 included in the PCR primers.The final construct is based on the binary vector pBin19 (Bevan, NuclAcid Res 12, 8711-8721 (1984)). The resulting plasmid contained the rolCpromoter and the octopine synthase polyadenylation signal (Gielen etal., EMBO J 3, 835-846 1984)).

1.2. Construction of Plasmid pBIN-IN8 (FIG. 1)

To obtain a truncated version of the yeast Suc 2 gene PCR using theoligonucleotides (SEQ ID NOS.: 9 and 10) 5′-Suc2d(GAGCTGCAGATGGCAAAGCAAACTAGCGATAGACCTTTGGTCACA) and 3′-Suc2d(GAGACTAGTTTATAACCTCTATTTTACTTCCCTTACTTGGAA) was applied to amplify theinvertase gene from plasmid PI-3-INV (von Schaewen et al. EMBO J 93033-3044, (1990)). The PCR product was digested with PstI/SpeI andcloned into the PstI/XbaI sites of plasmid YIP128A1 yielding plasmid181A1-INV (Riesmeier et al., EMBO J. 11 4705-13 (1992)). To obtain BamHIsites at both ends of the invertase gene plasmid 181A1-INV was digestedwith PstI/BamHI and the invertase fragment was ligated into vectorpBlueSK-yielding plasmid pBlue-Suc2A. Subsequently plasmid pBlue-Suc2Awas digested with SpeI/EcoRV, blunt ended with DNA polymerase and clonedinto the SmaI site of pBlueSK-yielding plasmid pBlue-Suc2B. Usingplasmid pBlue-Suc2B the invertase gene was isolated as a BamHI fragmentand cloned into the BamHI site of plasmid pBIN-RolC. The resultingplasmid (pBIN-IN8) contained the Suc2 gene (Nucleotide 849 to 2393)between the rolC promoter and the octopine synthase polyadenylationsignal (Gielen et al., EMBO J. 3, 835-46, 1984).

1.3. Transformation of Construct pBIN-IN8

Agrobacterium tumefaciens strain C58C1 containing pGV2260 (Deblaere etal., Nuc. Acid Res. 13, 4777-4788 (1989)) was transformed by directtransformation of variety Desiree by plasmid pBIN-IN8 as described byHöfgen and Willmitzer (Nucl Acid Res. 16, 9877 (1988)). Potatotransformation was achieved following the protocol of Rocha-Sosa et al.(EMBO J. 8, 23-29 (1989)). Primary screening for increased invertaseactivity was done in midribs of tissue-culture-grown regenerated plants.Three lines (DIN-87, 90 and 30) out of 75 independent transformants wereselected for further analysis. For a detailed analysis, ten replicatesof each preselected transformant were transferred into the green housefor tuber production.

1.4. Invertase Activity

Invertase assay. Plant tissue, quickly frozen in liquid nitrogen, washomogenised in extraction buffer (50 mM4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (Hepes)-KOH, pH 7.4;5 mM MgCl₂; 1 mM EDTA; 1 mM ethylene glycol-bis(b-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA); 1 mMphenyl-methylsulfonyl-fluoride (PMSF); 5 mM dithiothreitol (DTT); 0.1%Triton X-100, 10% glycerol) and centrifuged (5 min, 4° C., 9000 g,Biofuge 13; Heraeus, Hanau, Germany). For assaying neutral invertase thereaction mixture contained 20 mM Hepes-KOH pH 7.5, 100 mM sucrose and10-30 μl of the protein extract in a final volume of 100 μl. Incubationwas carried out at 30° C. for 30-60 minutes and stopped at 95° C. for 3minutes. Blanks had the same reaction mixture but were heat inactivatedwithout incubation. The determination of glucose and fructose was asdescribed in Stitt et al. (Methods Enzymol. 174, 518-522 (1989)). Forassaying soluble acid invertase the reaction mixture contained 20 mMsodium acetate pH 4.7, 100 mM sucrose and 10-30 μl of the proteinextract in a final volume of 100 μl. Incubation was carried out at 30°C. for 30-90 minutes. To neutralise the reaction mixture before stoppingthe reaction at 95° C. for 3 minutes 10 μl of 1 M sodium phosphate pH7.2 was added. Blanks had the same reaction mixture but were heatinactivated without incubation.

Following harvest tubers of transformed and untransformed potato plantswere stored for 5 months at 20° C. Subsequently neutral and acidicinvertase activity was determined in tuber slices. The result is shownin Table 1.

TABLE 1 Invertase activity in potato tubers stored for 5 month at 20° C.genotype neutral invertase soluble acidic invertase Control  32.7 ± 4.2 18.0 ± 1.3 DIN-87 115.3 ± 6.1 141.2 ± 8.8 DIN-90  93.9 ± 4.7 126.0 ±6.2 DIN-30 121.5 ± 8.4 174.8 ± 16.5

Mean values are given ± standard deviation (n=4). Invertase activity ispresented in nmol gFW⁻¹ min⁻¹. Control is wild type Desiree.

1.5. Impact of Invertase on Sugar Accumulation in Potato Tubers

Determination of soluble sugars. Tubers were harvested and tuber slices(60-70 mg fresh weight, 0.1 cm³ average volume) were immediately frozenin liquid nitrogen. The slices were extracted with 1 ml 80% ethanol (10mM Hepes-KOH, pH 7.4) at 80° C. for 1-2 h. The supernatant was used forthe determination of glucose, fructose and sucrose as described in Stittet al. (1989). The pellet was extracted a second time, washed in water,and dried. Determination of starch content was done using a starchdetermination kit (Boehringer Mannheim). The results are shown in Table2.

TABLE 2 Carbohydrate composition of potato tubers expressing cytosolicyeast invertase under control of the RolC promoter. Genotype FructoseGlucose Sucrose Starch Control 0.9 ± 0.1 6.2 ± 0.2 8.7 ± 0.4 652 ± 15DIN-30 0.3 ± 0.1 8.7 ± 1.0 2.1 ± 0.2 604 ± 19 DIN-87 0.8 ± 0.1 6.5 ± 0.43.1 ± 0.2 753 ± 26 DIN-90 0.8 ± 0.01 3.1 ± 0.6 3.5 ± 0.1 903 ± 39

Mean values are given±standard error (n=12, control; n=4, transgenic).Sugar content is presented as μmol hexoses gFW⁻¹. Control is wild typeDesiree

1.6. Yield

Potato plants were grown in a greenhouse at 60% relative humidity in a16 h light (22° C.) and 8 h dark (15° C.) cycle (irradiance 300 μmol m⁻²s⁻¹). To estimate the impact of phloem-specific cytosolic yeast-derivedinvertase on tuber fresh weight and tuber number ten plants eachgenotype were cultivated in 21 pots. As shown in Table 3, total freshweight and tuber number of the transgenic plants is indistinguishablefrom wildtype.

TABLE 3 Tuber yield of invertase expressing potato plants Genotype Tuberfresh weight Tuber number Control 118.3 ± 1.1  15 ± 0.01 DIN-87 116.5 ±6.1 11 ± 1.9 DIN-90 121.1 ± 0.2 12 ± 1.9 DIN-30   106 ± 10.5 13.5 ±2.8  

Mean values are given±standard deviation (n=10). Tuber fresh weight ispresented in g. Control is wild type Desiree

1.7. Sprout Inhibition of Transgenic Plants

To investigate the impact of phloem-specific cytosolic invertase ontuber sprouting harvested tubers of transformed and wildtype plants werestored for a prolonged time in the dark at room temperature. WildtypeDesiree tubers started to sprout after 5 to 6 months whereas tubers oftransgenic plants did not show any visible sign of sprouting. Even afterone year of storage tubers of transgenic plants did not develop anyvital sprout (FIG. 3). Thus, expression of phloem-specific invertaseleads to a complete inhibition of potato tuber sprouting.

2. Inhibition of Tuber Sprouting Via Expression of E. coli InorganicPyrophosphatase

2.1. Construction of Plasmid PPA-2 and Potato Transformation (FIG. 2)

The 1600 bp promoter fragment of the STLS-1 gene (Eckes et al., Mol.Gen. Genet. 205, 14-22 (1986)) was isolated as a EcoRI-BamHI fragmentfrom plasmid 1600-CAT (Stockhaus et al., 1987). After removal of theoverlapping nucleotides the fragment was cloned into the blunted EcoRIsite of the chimeric ppa gene described in Sonnewald (Plant J. 2,571-581 (1992)). The final construct containing the STLS-1promoter/enhancer, the 35S CaMV promoter, the TMV-U1 translationalenhancer, the E. coli ppa coding region and the octopine synthasepolyadenylation signal was cloned as a EcoRI-HindIII fragment into thebinary vector Bin19 (Bevan, 1984 J. loc cit). Direct transformation ofAgrobacterium tumefaciens strain C58C.1:pGV2260 was done as described byHöfgen and Willmitzer (Nucl Acid Res. 16 9877 (1988)). Potatotransformation using Agrobacterium-mediated gene transfer was performedas described by Rocha-Sosa et al. (EMBO J. 8 23-29 (1989)).

Following Agrobacterium mediated gene transfer forty independenttransformed plants were analysed for the presence of the PPase proteinusing inmmunoblotting. Three plants (PPaII-2, 3 and 5) with the highestamount of PPase protein were selected for further analysis. To comparethe promoter strength of the chimeric 35S CaMV promoter (PPaII) and theunmodified 35S CaMV promoter (PPaI) protein extracts from potato tuberswere analysed by western blotting. As shown in FIG. 4, the amount of thePPase protein, detectable in protein extracts from growing PPaII tubers,is significantly higher as compared to the PPaI control. The sameresults were obtained in tubers stored for three and twelve months atroom temperature. This analysis compared the highest expressing linesfrom the PPaI and PPaII populations where 70 independent transformantswere selected for PPaI and 40 for PPaII. It is clear from this analysisthat the STL1 promoter fragment enhances tuber expression of inorganicpyrophosphatase. The expression of the E. coli inorganic pyrophosphatasewas paralleled by an increase in pyrophosphatase activity measured inprotein extracts from PPaII tubers (Table 4). Depending on the amount ofpyrophosphatase activity the pyrophosphate content decreased up totwo-fold (Table 4).

TABLE 4 Elevated cytosolic inorganic pyrophosphatase leads to reducedPPi accumulation in tubers of PPaII transformants. Pyrophosphataseactivity Pyrophosphate Genotype [μmol g FW⁻¹ minute⁻¹] [nmol g FW⁻¹]Control 3600 ± 410 2.4 ± 0.2 PPaII-2 5600 ± 150 1.4 ± 0.2 PPaII-3 6200 ±220 1.2 ± 0.3 PPaII-5 8500 ± 220 1.1 ± 0.1

Tubers were harvested from plants grown for 150 days in the greenhouse.The results are mean values±SD (n=3 for wildtype and n=4 for transgenicplants) of three tubers from three different wildtype plants and two tofour tubers each PPaII plant.

2.2. Immunoblot Analysis

Following separation on 12.5% SDS polyacrylamide gels (Laemmli, 1970),proteins were transferred onto nitrocellulose membranes (Millipore,Bradford, Mass., USA) using a semi-dry electroblotting apparatus(Multiphore II; LKB, Bromma, Sweden). Incubation with anti-PPasepolyclonal antibodies (Lerchl et al., Plant Cell 7 259-270 (1995)) in a1:1000 dilution was for 90 minutes at room temperature. Immunodetectionof the antigen was done using the biotin-streptavidin system fromAmersham Buchler with rabbit biotinylated species-specific wholeantibodies (from donkey) and streptavidin-biotinylated horse-radishperoxidase.

2.3. Pyrophosphatase Activity Assay

To measure pyrophosphatase (PPase) activity 100-200 mg potato tuberslices were homogenised in 0.5 ml 100 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH (pH 7.5),2 mM Mg2Cl, 1 mM EDTA, 1 mM EGTA, 5 mM mercaptoethanol. Aftercentrifugation (10 minutes, 13.000 rpm at 4° C.) 20 μl of thesupernatant was assayed in 160 μl 50 mM Tris-HCL (pH 8.0), 16 mM MgSO4and 100 mM KCl for PPase activity. Following addition of 20 μl 50 mMNaPPi the reaction was carried out for 20 minutes at 30° C. The reactionwas stopped by addition of 20 μl 1 M citrate and the release ofinorganic phosphate was assayed as in Heinonen and Lathi (Anal Biochem113, 313-317 (1981)). The assay was linear with time and amount ofextract.

2.4. Determination of Inorganic Pyrophosphate in Tuber Tissue

To measure pyrophosphate 200-300 mg of tuber tissue was frozen in liquidnitrogen. Frozen material was subsequently homogenised to a fine powderin liquid nitrogen in a mortar standing on powdered dry ice (solid CO₂).A 15 ml aliquot of 16% trichloroacetic acid (TCA) in diethylether (v/v),precooled to the temperature of dry ice, was added and the samplefurther homogenised. After leaving the homogenate for 20 minutes on dryice, 0.8 ml of 16% TCA in water (v/v) containing 5 mM NaF was added. Themixture was warmed to 4° C. and left for 3 hours. Subsequently thehomogenate was extracted four times with diethylether and neutralisedwith KOH/triethanolamine as in Weiner et al. (Biochem Biophys Acta 893,13-21 (1987)). All mortars and materials were prewashed for 12 hours in2 N HCl, and pseudoextracts were prepared in parallel to check that thereagents and apparatus were not contaminated with pyrophosphate. eforeassaying for pyrophosphate content 400 μl of extract was added to 400 μlof cation exchanger (Serva, Heidelberg, FRG; Dowex AG 50×8, 100-200Mesh, preequilibrated with 2 N HCl, brought to pH 5 with water, and thendried for 12 hours at room temperature), mixed for 20 seconds, andcentrifuged to remove compounds in the extract which interfere with themetabolite assay. Pyrophosphate was assayed photometrically as in Weineret al. (1987). The reliability of the extraction and assay was checkedby adding a small representative amount (two- to threefold theendogenous content) of pyrophosphate to the plant material in the killedmixture of TCA and diethylether.

2.5. Yield

Potato plants were grown in a greenhouse at 60% relative humidity in a16 h light (22° C.) and 8 h dark (15° C.) cycle (irradiance 300 μmol m⁻²s⁻¹). To estimate the impact of the E. coli inorganic pyrophosphatase ontuber fresh weight and tuber number five plants each genotype werecultivated in 2 l pots. Total tuber fresh weight of PPaII plants wasunaltered as compared to wildtype controls (Table 5).

Table 5: Influence of E. coli inorganic pyrophosphatase on potato tuberdevelopment. The tubers were harvested from plants which had beengrowing in the green house for 150 days. The results are means of fiveindividual plants each genotype.

TABLE 5 Influence of E. coli inorganic pyrophosphatase on potato tuberdevelopment. The tubers were harvested from plants which had beengrowing in the green house for 150 days. The results are means of fiveindividual plants each genotype. Genotype Tuber fresh weight [g] Tubernumber Control 89-148 12 ± 0.6 PPaII-3 99-139 19 ± 1.7 PPaII-5 110-167 21 ± 4.0 PPaII-2 89-192 19 ± 0.5

of expression in the PPaI transgenic plants was not sufficient toprevent sprouting. 2.6. Sprout inhibition of transgenic plants.

Tubers harvested from wildtype plants started to sprout after five tosix months of storage, whereas PPaII tubers did not develop any visiblesprout (FIG. 5). While sprout development of wildtype tubers continued,there was still no indication of sprouting in PPaII tubers after twelvemonths of storage. Even after a prolonged storage of two years, PPaIItubers did not sprout. Treatment of potato tubers with gibberellic acid,ethephon, higher- and lower temperatures or light did not inducesprouting of PPaII tubers.

II. Exemplification of Inducible Gene Expression in Potato Tubers 3.Ethanol Inducible Gene Expression

3.1. Construction of Plasmid Alc:GUS

The source of the GUS gene was the pUC based plasmid pJIT166 (FIG. 6). Afragment containing the GUS coding region and CaMV35S terminator, frompJIT166 was cloned into pACN/pUC vector using SalI and BglII. BglII cutsthree times in the CaMV35S terminator. The first cut occurs 250 basesbeyond the end of the GUS gene. Although this only takes a small part ofthe terminator the fragment contains all necessary sequences requiredfor the termination of transcription. The SalI-BglII digest of pJIT166yielded a 1.8 kbp fragment containing the GUS gene plus the truncatedCaMV35S terminator. This fragment was cloned into pACN/pUC digested withSalI and BglII to remove the CAT gene and the nos terminator leaving aSalI overhang at the 5′ end behind the alcA promoter and a BglIIoverhang at the 3′ end of the linearised vector. The fragment containingthe GUS gene and the CaMV35S terminator was ligated into the linearisedpUC vector containing the alcA promoter using standard protocols. Thefinal step in the cloning procedure was to clone the alcA-GUS-35Stfragment into pSRN-ACN/BinN19, in place of the alcA-CAT-nos fragment.The resulting Bin19 vector would then contain all the components of thealc regulon but with the GUS reporter. The alcA-CAT-nos fragment wasexcised from pSRN-ACN/Bin19 vector with a HindIII digestion. Theremaining 16.1 kbp fragment, which is the Bin19 vector still with the35S-alcR-nos region, was extracted from the gel by electro-elution. ThealcA-GUS-35St fragment was then excised with a HindIII XmnI doubledigest of pAGS/pUC. The restriction enzyme XmnI cuts approximately 850bp off the pUC19 vector giving separation and allowing the removal ofthe alcA-GUS-35St fragment. The alcA-GUS-35St fragment was then clonedinto the vacant HindIII site in pSRN/Bin19. The fragment was orientatedusing restriction mapping and then sequenced to confirm that theycontained the correct sequences. A map of plasmid AlcR/AGUS is providedin FIG. 7.

3.2. Transformation of Construct

Direct transformation of Agrobacterium tumefaciens strain C58C1:pGV2260was done as described by Höfgen and Willmitzer (1988)(J. loc cit.).Potato transformation (Solara) using Agrobacterium-mediated genetransfer was performed as described by Rocha-Sosa et al. (1989) (J. loccit).

Following Agrobacterium mediated gene transfer 100 independenttransformed plants were selected. To test inducibility of the GUSactivity shoots of transgenic plants were duplicated in tissue culture.Following root formation one set of plants was transferred into thegreenhouse. Two weeks after transfer into the greenhouse ethanolinducibility was assayed by adding 50 ml of a 5% ethanol solution to theroot system of the potato plants. Subsequently GUS activity wasvisualised using the histochemical detection system. Following ethanolinduction GUS activity was visible in all tissues tested (sink- andsource leaves, stem, roots and stolons). As shown in FIG. 8 GUS activitywas highly inducible in developing and mature tubers. There was nodetectable GUS activity in any organ in uninduced potato plants.

In order to investigate the sensitivity of the Alc-switch to ethanolvapour an experimental system was used where an Alc-CAT(chloramphenicalacetyl transferase) tobacco plant (CaMV35S-AlcR-nos, AlcA-CAT-nos;Caddick et al., 1998) was enclosed in a sealed container with a pot ofethanol of a particular concentration to act as a source of ethanolvapour. Headspace and leaf samples were taken after 24 hours. Absoluteamounts of ethanol in the headspace samples was quantified by relatingthe ethanol peak area obtained after injection using a gas-tight syringeinto a gas chromatography machine with a mass spectrometry detector tothat with an ethanol standard solution. Total CAT expression levels inleaves were determined by CAT ELISA. CAT expression in tobacco plantsenclosed with ethanol solutions of 5, 1, 0.1 and 0.05% were relativelyconstant but dropped dramatically with 0.01, 0.005 and 0.001% ethanolsolutions (see FIG. 28). Relating the levels of CAT activity to ethanolvapour concentrations in the container, the threshold of Alc-switchactivation was seen at an ethanol concentration of between 72 and 21ng/ml air.

To further study the inducibility of GUS in stored potato tubers fourGUS positive transgenic lines were selected for a detailed analysis.After multiplication in tissue culture 5 plants of each genotype weretransferred to the green house for tuber production. Following harvesttubers were placed in a sealed glass container containing 3MM papersoaked with a 5% ethanol solution.

To prove that ethanol induction would be efficient throughout the wholepotato tuber slices were taken at different times following ethanolinduction and GUS activity was visualised using the histochemicaldetection method. As shown in FIG. 9 homogenous induction of GUSactivity was found in intact potato tubers.

The use of ethanol vapour to activate the Alc-switch was investigated inAlc-GUS potato tubers (CaMV35S-AlcR-nos, AlcA-GUS-nos). The kinetics ofGUS RNA transcript accumulation was determined by northern analysis.Potato tubers were enclosed with an ethanol source for 3, 6, 9, 12, 24,48 hours and 1 week time points, the ethanol source removed and samplessubsequently taken at 2, 3 and 4 week time-points. By varying theconcentration of ethanol used for induction in the enclosed system, thetimecourse of GUS transcript accumulation can be altered. Using 8 ml ofabsolute ethanol in a 40 liter container low levels of GUS transcriptcan first be detected at 6 h in the outer 1-3 mm below the tuber skinand at 12 h 3 mm or more below the skin surface (see FIG. 29). Maximallevels of transcript were detected at 24 h with transcript persistinguntil 4 weeks. In contrast, using a 5% ethanol solution to generate alower ethanol vapour concentration transcript is first detected at 1week. By keeping the a constant ethanol source GUS transcript wasdetected at high levels through-out the course of the experiment (lasttime-point 3 months) (see FIG. 30).

An extension of these ethanol vapour studies was to investigateAlc-switch induction in tomato fruit. Using a 5% ethanol solutionenclosed in a 2.6 l container with Alc-GUS tomato fruit(CaMV35S-AlcR-nos, AlcA-GUS-nos), significant GUS staining was observedin the walls of the pericarp originating from the stig in fruit after 4weeks of ethanol exposure. Tomato fruit were sliced, washed briefly in50 mM sodium phosphate buffer, pH. 7.0 and incubated in staining buffer(50 mM sodium phosphate buffer, pH. 7.0, 50 uM potassium ferricyanide,50 uM potassium ferrocyanide, 2% triton X100, 20% methanol and 1 mM5-bromo-4-chloro-3-indolyl-B-D-glucoronide) as required. Staining wasstopped by performing 100% and 70% ethanol washes and the fruit slicesstored in 70% ethanol at 4° C.

3.3. Fluorometric Determination of GUS Activity

The fluorometric determination of GUS activity was carried out asfollows: Tuber slices harvested after the indicated times followingethanol induction were frozen in liquid nitrogen. Subsequently tubertissue was homogenised in 50 mM NaHPO4 (pH 7.0), 10 mM mercaptoethanol,10 mM EDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100. Thehomogenate was centrifuged for 10 minutes at 13.000 rpm at 4° C., thecleared supernatant was collected and used for the determination ofprotein content and GUS activity. For fluorometric detection 20 μl ofextract (diluted to a proper concentration) was added to 480 μl GUSassay buffer (2 mM MUG in extraction buffer) and incubated for 30minutes at 37° C. Thereafter 50 μl of the reaction mixture wastransferred to 1950 μl stop solution. The fluorimetric signal of eachsample was determined with a TKO 100 mini-fluorometer (excitation at 365nm, emission at 455 nm). From the initial slope of the curve obtained byplotting the fluorometric value against time enzyme activity wascalculated. Heat inactivated extract served as controls. The activityvalues were normalised to the protein concentration of each extract.

3.4. Histochemical Detection of GUS Activity

For histochemical detection of GUS activity tissue samples wereincubated in X-gluc buffer (25 mM sodium phosphate buffer (pH 7.2), 25mM potassium phosphate (pH 7.2), 0.1% Triton X-100, 1 mM X-gluc). Briefvacuum infiltration (30 seconds) was used to support penetration of thesubstrate into the plant tissue. Subsequently the material was incubatedat 37° C. for 3 to 24 hours and rinsed with water before photography.Photosynthetic tissue were bleached with ethanol. Microscopic analysiswas performed using an Wild Makroskop M420 equipped with a Wild MPS46photoautomat.

III. Safener Inducible Gene Expression in Potato Plants

4.1. Construction of GST:GUS Plasmid

Standard recombinant DNA methods were adopted in the construction ofplasmid vectors. A reporter gene construct containing a GST-27 3.8 kbEcoRI-Nde I 5′ flanking region from pG1E7 was blunted ended and ligatedinto the Sma I site of the Agrobacterium Ti vector pBI101. The Nde Isite, which lies at the predicted translation start codon of GST-27 wasdestroyed after blunting. This formed a convenient point for fusion withthe E coli UidA gene, encoding b-glucuronidase (GUS) in pBI101. Thestructure of the resultant chimeric reporter gene construct pGSTTAK wasverified by restriction and sequence analysis. A map of plasmid pGSTTAKis provided in FIG. 10.

4.2. Transformation of Construct and Test of Inducibility

Using plasmid pGST::GUS direct transformation of Agrobacteriumtumefaciens strain C58C1:pGV2260 was done as described by Höfgen andWillmitzer (1988) (J. loc cit). Potato transformation (Solara) usingAgrobacterium-mediated gene transfer was performed as described byRocha-Sosa et al. (1989) (J. loc cit).

Following Agrobacterium mediated gene transfer 100 kanamycin resistantregenerated shoots were selected. Safener inducibility was tested bytransferring stem cuttings of GST::GUS transformed potato plants on MSmedium containing 0, 0.4, 2.0 and 10% R-25788 (final concentration).Following cultivation for 14 days fully developed leaves were harvestedand GUS activity determined. As shown in FIG. 11, a 3 to 20 foldincrease in GUS activity could be obtained following safener induction.

IV. Inducible Repression of the Expression of Target Genes 5. InducibleCo-suppression

5.1. Construction of Chimeric Gene for Inducible FNR Co-suppression

To achieve ethanol inducible co-suppression of NADP-ferredoxineoxidoreductase (FNR) a ca. 450 base pair 3′-fragment (Seq. 1) of atobacco FNR cDNA was fused to the alcA promoter in the sense orientationyielding plasmid SQ03. The cloning strategy is illustrated in FIG. 12.

Direct transformation of Agrobacterium tumefaciens strain C58C1:pGV2260was done as described by Höfgen and Willmitzer (1988) (J. loc cit).Tobacco transformation (Samsun) using Agrobacterium—mediated genetransfer was performed as described by Rosahl et al. (EMBO J 6, 23-29,(1987)).

Following Agrobacterium mediated gene transfer 100 independenttransformed plants were selected.

Seq. 1 (SEQ ID NO.: 1): Total number of bases 423

TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTCCCAAAAA ATGAAATTAAAATTTCAAAG GAAAAATTTA CCTATCTACA TGGATGCAGG GGGAGAGAAG CATAAAGTTGGCTCATATTT GTACAAAGAA AAGTAAAAAT ATTTAGTAGA CTTCAACATT CCATTGCTCTGCCTTCTTCA ATTGCTTCTT GTAGTCCGCC CAGACAATAC CATCTCTTTC AGCAAGAGCAGACATAATTT CATCAATTCC CTGCTCCATG CCCTTGAGTC CACACATGTA GATGAAGGTGTTGTCTTTTT GGAGCAAAGT CCATAGTTCT TCAGCATATT GAGCCATTCT GGTTTGAATGTACATCTTTT CACCCTTTCC GTTCGTTTGC TCTCTGCTCA CAGCAAAGTC CAATCTGAAG TTT-3′

V. Inducible Antisense Repression of Heterologous Genes

As described in section I expression of cytosolic invertase andinorganic pyrophosphatase can lead to a non-sprouting phenotype whensuitable promoters are used to drive the expression of the respectivegene. To achieve inducible reversion of the non-sprouting phenotype astrategy for the inducible antisense of the heterologous gene wasapplied.

6. Inducible Antisense Repression of Pyrophosphatase Expression

6.1. Construction of Plasmid SQ01

As shown in FIG. 5 high level expression of E. coli inorganicpyrophosphatase leads to a non-sprouting phenotype of harvested potatotubers. To chemically control the expression of E. coli inorganicpyrophosphatase plasmid SQ01 was designed. The plasmid contains threechimeric genes: (a) the alcR gene under control of the 35S CaMVpromoter, (b) the ppa gene under control of the enhanced 35S CaMVpromoter and (c) the ppa gene in the antisense orientation under controlof the alcA promoter. The construction of plasmid SQ01 is shown in FIG.13.

6.2. Plant Transformation

Direct transformation of Agrobacterium tumefaciens strain C58C1:pGV2260was done as described by Höfgen and Willmitzer (1988) (J. loc cit).Potato transformation (Solara) and tobacco (Samsun) usingAgrobacterium—mediated gene transfer was performed as described byRocha-Sosa et al. (1989) (J. loc cit) and as described by Rosahl et al.(1987) (J. loc cit).

Following Agrobacterium mediated gene transfer 100 independenttransformed plants were selected.

6.3. Immunological Detection of ppa

The successful transformation was tested by the immunological detectionof the E. coli pyrophosphatase protein in leaf extracts of tissueculture grown potato plants. Based on the initial screening 15independent transgenic plants could be identified. Following duplicationin tissue culture pyrophosphates expressing transgenic potato plantswere transferred into the green house for tuber formation.

7. Inducible Antisense Repression of Invertase Expression

7.1. Construction of Plasmid SQ02

As shown in FIG. 3 phloem-specific expression of cytosolic yeast-derivedinvertase leads to a non-sprouting phenotype of harvested potato tubers.To chemically control the expression of yeast invertase plasmid SQ02 wascreated. The plasmid contains three chimeric genes: (a) the alcR geneunder control of the 35S CaMV promoter, (b) the truncated suc2 geneencoding the mature invertase protein under control of the rolC promoterand (c) the suc2 gene in the antisense orientation under control of thealcA promoter. The construction of plasmid SQ02 is shown in FIG. 14.

7.2. Plant Transformation

Direct transformation of Agrobacterium tumefaciens strain C58C1:pGV2260was done as described by Höfgen and Willmitzer (1988) (J. loc cit).Potato transformation (Solara) and tobacco (Samsun) usingAgrobacterium-mediated gene transfer was performed as described byRocha-Sosa et al. (1989)(J. loc cit) and as described by Rosahl et al.(1987) (J. loc cit).

Following Agrobacterium mediated gene transfer 100 independenttransformed plants were selected.

7.3. Invertase Activity

The successful transformation was tested by the detection of invertaseactivity in SDS PAA-gels as described in von Schaewen et al. (EMBO J. 9,3033-3044 (1990)). To this end protein extracts were prepared frommidribs of tissue culture grown potato plants. Following separation ofthe protein extracts in 12.5% SDS PAA gels the gel was washed in 100 mMNa-Acetate buffer pH 5.0 for 30 minutes. Subsequently the gel wasincubated in 100 mM Na-Acetate buffer containing 100 mM Sucrose at 37°C. for 1 hour. After a brief wash with distilled water invertaseactivity was visualised via the detection of liberated reducing sugars(glucose and fructose). Hexoses were detected by boiling the gel in 0.1%2,3,5-Triphenyltetrazoliumchlorid in 0.5N NaOH for 2-5 minutes.Invertase activity became visible due to the formation of an intense redcolour. Based on the initial screening 18 independent transgenic plantscould be identified. Following duplication in tissue culture invertaseexpressing transgenic potato plants were transferred into the greenhouse for tuber formation.

V1. Use of Operator/Repressor System to Repress Heterologous Genes 8.Use of Lac Operator/Repressor System with the rolC Promoter, theYeast-derived Invertase Gene and the Alc Switch

8.1. Cloning of lacI into ALC Switch Binary Vector

As can be seen from FIG. 22, the lacI-nos region was excised from a35S-LacI-nos plasmid with BamH1/HindIII and cloned into a BamH1/HindIIIdigested pMSC2 vector. This vector has a Pst1 site a few bases 5′ to theBamH1 site, so a Pst1 digest removed the lacI coding region. This wasthen cloned into a pst1 digested pACN vector (AlcA-cat-nos), replacingthe AlcA gene with the lacI gene, to give a pALN vector (AlcA-lacI-nos).The ALN region was removed from this with a HindIII digest and clonedinto a HindIII digested binary SRNACN (35S-AlcR-Nos-AlcA-Cat-Nos)vector, replacing the ACN with the ALN cassette.

8.2. Cloning the lacI Operator into RolC-invertase

Two oligonucleotides (SEQ ID NOS.: 11 and 12) were synthesized withBamHI and Asp718 restriction sites SC24:TTGGTACCAATTGTGAGCGCTCACAATTGGATCCTT SC25:AAGGATCCAATTGTGAGCGCTCACAATTGGTACCAA. 10 uM of both oligonucleotideswere annealed by boiling in a water bath in the presence of 20 mMTris.Cl (pH8.4) 50 mM KCl and 1.5 mM MgCl₂ for 5 minutes before coolingdown to 30° C. over approximately one hour, followed by 5 minutes onice. The annealed oligonucleotides were digested with BamH1 and Asp718,and the restriction enzymes inactivated by phenol extraction and ethanolprecipitation. The fragments were ligated into BamH1 and Asp718 cutpUC19 to give pUC-lacO. The plasmid was confirmed by sequencing. The OCSterminator was removed from the BINAR plasmid and cloned into the SalIand HindIII restriction sites of plasmid pUC-lacO creating the plasmidpUC-lacO-ocs. The RolC promoter was inserted with EcoRI and Asp718(KpnI) to give the plasmid pUC-RolC-lacO-ocs. The yeast derivedinvertase was inserted into the BamHI site of pUC-RolC-lacO-ocsresulting in the plasmid pUC-RolC-lacO-INV-ocs. FIG. 21 shows thecloning strategy for this plasmid.

8.3. Ligation of Above Two Components to Give the Final Binary Vector

The RolCopINVocs cassette is on a HindIII fragment (using the 865bp RolCpromoter) and was ligated to a HindIII digested binary SRNAlacI in athree-way ligation, to give the final construct of35S-AlcR-nos-AlcA-lacI-nos-RolC-op-invertase-ocs.

VII. Additional Targets

Based on known biochemical steps involved in potato tuber sprouting wehave identified several additional targets which may be used to creategenetically engineered non-sprouting potato tubers. Besides others,respiratory enzymes or membrane proteins involved in the mitochondrialexport of metabolites are promising. One of these candidates is themitochondrial ATP/ADP translocator and a second malate oxoglutaratetranslocator.

9. Genes Involved in Mitochondrial Function

9.1. Cloning of ATP/ADP Translocator (ANT) and Construction of aChimeric Antisense Gene

Based on a published sequence of potato ADP/ATP translocator (Emmermannet al. (1991) Curr. Genet. 20, 405-410) oligonucleotides were designedto allow PCR-amplification of an internal ANT-fragment (see FIG. 15).The following PCR-primers (SEQ ID NOS: 13 and 14) were used: 5′-ANTprimer: 5′-AACGGATCCATGGCAGATATGAACCAGC-3′; 3′-ANT primer: 5′-TTGGATCCTTACAACACACCCGCCCAGGC-3′. To optimise subsequent cloning of theANT-fragment into plant expression vectors BamHI sites were included inboth PCR primers. As template reverse transcript mRNA isolated fromgrowing potato tubers was used. RNA isolation was done according toLogemann et al. (1987; Anal. Biochem., 163, 16-20). Single strand cDNAwas synthesised using M-MLV superscript reverse transcriptase accordingto the instructions of the manufacturer (Gibco, BRL). The temperatureprofile of the PCR cycle (40 cycles) was as follows: 1 min at 95° C., 1min at 45° C., and 2 min at 72° C. The amplified DNA was cloned into thePCR vector pCR1000™ (Invitrogen, Norwalk, Conn.). To exclude mutationsof the amplified DNA during the PCR cycles, the clone was sequencedusing the dideoxy method. The 1120-bp ANT fragment was subsequentlycloned into a plant expression cassette pBINAR (Höfgen and WillmitzerPlant Sci. 66 221-230 (1990)) in the antisense orientation (FIG. 15).

9.2 Cloning of Mitochondrial Oxoglutarate Translocator (MOT)

A cDNA fragment encoding MOT was isolated using the methods described inSection 9.1. Northern analysis on FIG. 17 shows the MOT mRNA expressionis highest when RNA is extracted immediately below the tuber sprout.This region corresponds to high metabolic activity. An antisensedownregulation construct was prepared by amplifying the MOT fragmentessentially as described above in section 8.1 for ANT but using theprimers shown in FIG. 18. The BamHI/SalI PCR fragment was cloned intoBam/Sal cut pBluescript SK. To exclude mutations of the amplified DNAduring the PCR cycles, the clone was sequenced using the dideoxy method.An Asp718/BamHI fragment was excised from the pBluescript vectordescribed and cloned into BamHI/Asp718 cut pBinAR. This yielded a planttransformation cassette containing the 35S CaMV promoter driving MOT inan antisense orientation.

9.3. Transformation

Direct transformation of Agrobacterium tumefaciens strain C58C1:pGV2260was done as described by Höfgen and Willmitzer (1988) (J. loc cit.).Potato transformation using Agrobacterium—mediated gene transfer wasperformed as described by Rocha-Sosa et al. (1989)(J. loc cit).

Following Agrobacterium mediated gene transfer 70 independenttransformed plants were selected.

10. Genes Induced During Potato Tuber Storage

10.1. Isolation of Genes Induced During Tuber Storage

10.1.1. Differential Display

To gain insight into molecular changes occurring during the transitionof growing to sprouting tubers the differential display technique wasused. To this end total RNA was isolated from growing and stored potatotubers (Desiree). Following DNaseI digestion 5 μg of total RNA wasreverse transcript using M-MLV superscript reverse transcriptase (Gibco,BRL) yielding single strand cDNA templates. Subsequently, PCRamplification of the prepared cDNA templates was carried out in thepresence of (α-³⁵S)dATP using oligo-d(T)11-XN and 100 different RAPDprimers. The use of the following RAPD-primers led to the isolation ofsource tuber-specific cDNA fragments (SEQ ID NOS.: 15 and 17):5′-AAGCGACCTG-3′; 5′-GTTGGTGGCT-3′; 5′-ACGGGACCTG-3′.

The temperature profile of the PCR cycle (40 cycles) was as follows: 30seconds at 94° C., 1 min at 42° C., and 30 seconds at 72° C. Theamplified DNA was denatured for 5 minutes in formamide buffer at 94° C.and loaded onto a PAA-gel (6% acrylamide, 0.3% bisacrylamide, 7 M ureain TBE buffer). Separation of the cDNA fragments was done at 1.75 KV,130 mA for 3 hours. Following separation the gel was dried at 80° C. andradioactive labelled cDNA fragments were visualised via autoradiographyusing Kodak X-OMAT X-ray films. Exposure time ranged from 2 to 5 days.Comparison between cDNA fragments amplified from growing or sproutingtuber templates allowed the detection of cDNA fragments beingexclusively present in sprouting potato tubers. Sprouting tuber-specificcDNA fragments were subsequently eluted from the PAAG and reamplifiedusing the respective PCR primers. The reamplified cDNA fragments weresubsequently cloned into the PGEMT vector (Promega). The size of theamplified cDNA fragments varied between 200 and 450 base pairs.

10.1.2. Northern Blot Analysis of Genes Induced During Tuber Storage

To verify that the isolated cDNA fragments are induced in stored potatotubers total RNA of growing and 1, 7, 14, 21, 30, 60, 90, 120, 150 and180 days stored potato tubers was isolated, separated in 1.5%formaldehyde (15% v/v) containing agarose gels and probed for thepresence of the respective transcripts following transfer of the RNAonto nylon membranes. As shown in FIG. 16 the transcripts of 4 isolatedclones (16-3, 10-1, AC4 and 16-8) accumulate during potato tuberstorage.

10.1.4. Construction of cDNA Library

To obtain full size cDNA clones encoding M-1-1, 16-3, 10-1, AC4 and 16-8a stored tuber-specific cDNA library was constructed. To this end polyARNA was isolated from potato tubers stored for 5 months at roomtemperature. cDNA synthesis was carried out using a cDNA synthesis kitfrom Pharmacia. Following adaptor ligation (EcoRI/NotI-adaptors) thecDNA was ligated into lambda ZAP II vectors following the instructionsof the manufacturer (Stratagene). In vitro packaging was carried outusing the Gigapack²II Gold packaging extract from Stratagene.

10.1.5. Isolation of cDNA Clones Encoding Stored Tuber-specific cDNAClones

Following amplification of the primary cDNA library 2×10⁵ Pfu (plaqueforming units) were screened for the presence of phages hybridising toM-1-1, 16-3, 10-1, AC4 and 16-8 PCR-fragments. In all cases severalindependent phages hybridising to the respective PCR probes wereisolated and restriction analysis following in vivo excision of theisolated clones was carried out. In four cases (M1-1, 16-3, 10-1 and16-8) full size cDNA clones could be obtained. After determination ofthe complete nucleotide sequences (Seq. 2 to 6) and FIG. 19 a homologysearch was carried out. Based on homologies clone 16-3 corresponds toubiquitin carboxyl-terminal hydrolase from human, Drosophila and yeast,clone 10-1 was found to be identical to the ADP-ribosylation factor 1from potato (belonging to the family of GTP-binding proteins) and clone16-8 has homologies to a auxin repressed protein of unknown functionfrom strawberries. No homology was found for clone AC4. Differentiallyexpressed clone M-1-1 encodes a protein which we have designated as MOTand which was found to have homology with bovine and human mitochondrial2-oxoglutarate carrier protein. The sequence comparison is provided inFIG. 20.

10.1.6. Nucleotide Sequence of Induced Clones

Seq. 2 (SEQ ID NO.: 2): 16-3 (homology to ubiquitin carboxyl-terminalhydrolase from human, Drosophila and yeast)

GGGCTGCAGGAATTCGAGGCCGCTAGAGAGAGTTAAAATAGAGGAAAGGAATCCATGGCGGAAAGCACAGGCTCTAAGAAGAGATGGCTTCCTCTTGAAGCTAACCCCGATGTCATGAATCAGTTTCTTTGGGGTCTTGGTGTTCCACCGAATGAGGCCGAGTGCTGTGATGTTTATGGGTTAGATGAAGAACTTCTGGAGATGGTGCCAAAGCCAGTGCTTGCTGTTTTATTTCTCTATCCTCTCACATCTCAGAGTGAAGAAGAGAGAATAAAGCAAGACAGCGAAACAAAGGTGCAGGATCCCAGTAGTACAGTTTACTACATGAAACAAACAGTGGGAAATGCATGCGGAACAATTGGCCTTCTTCATGCTATTGGGAATATCACCTCTCAGATAAAACTTACCGAGGGTTCATTCTTGGACAAGTTCTTTAAATCAACCTCAAGCATGGACCCAATGCAGCGTGCTTTGTTCCTTGAAAATGATAGGGAAATGGAAGTTGCTCATTCAGTGGCAGCCACTGCTGGTGATACTGAGGCTACCGACGATGTGAACGCTCATTTCATCTGCTTCACCTGTGTTGATGGACAACTCTATGAACTTGATGGAAGGAGGGCTGGACCTATTACACATGGCGCATCCTCTCCAAACAGCTTATTAAAGGATGCAGCCAGAGTTATCAAAAAGATAATCGAGAAAAATCCAGACTCAATCAACTTCAACGTTATTGCTATTTCCCAAAACGTTTAGGCCAATCTAGAGGCTTTTATCGATGAGATGGTTTAAACCAATTTTAGCTTTTCATGTTTCTGCCGTTTCCAGTACTATGTTTCTTCTTGTTTGCAATAAGTTACTTTTGAGAAAAAA

Seq. 3 (SEQ ID NO.: 3): 16-8 (homology to auxin-repressed protein fromstrawberry)

TGTTCTATCCCAGCGGACGCAGAATTTCCTTTTTTATTCTTCTCTTCTTCTCCCCTAAAACGTGAGCCGATTGGCTAACCTGCACCATGAGCTTACTTGACAAGCTCTGGGACGACACCGTTGCCGGTCCCCTGCCAGATAGTGGCCTCGGGAAACTCCGGAAGTATTCTACTTTTAGTCCGCGTTCAAATTCCGGCAAGGAATCAGAAGTTTCCACACCGAGATCCTTCACCGAGGAAGCAAGTGAGGACGTGGTGAAGGTGACGAGAAGTATCATGATAGTAAAGCCTTCCGGGAGTCAGAATAGAGATTCACCTCCAGTTTCTCCGGCCGGTACTACTCCTCCGGTATCTCCTTTTGCCCCTTCCGCTGGAAGAGAAGCATTTCGGTTCCGGCGGCGATCAGCGTCATTTGCATACGAGAATGCCAGTGGGGTTGGACCCAGAAGCCCTCGTCCTCCTTACGACCTGTGAGATATAGTCGGGTTCTCTTTTTTTGTTATCCCTCTTGAGGCGGTTGAATGTAGTATAGCTAGTCGACATACTCAACATGTTCCTGGTTGAGAGTGTTGTTTTGTGTGGTGTTTAATTTGTTTGCTTAATTTTGTAAATAGTGCAAGTGGTTCTTCATCTTGCGGATGTTGTGACGAAGGTTTAGCACAAGATGTAAGCGTCCAAGTTGGTCATGTATTCTGCTTTGTATTAAAAAAAAA

Seq. 4 (SEQ ID NO.: 4): 10-1 (ADP-RIBOSYLATION FACTOR 1 from potatobelonging to the family of GTP-binding proteins)

TGGACAATAGAGATCTACTGATTTCATCCTCTCTCATCGGCCGATCTTCGATTAACGGAGATGGGGCTGTCTTTCACTAAACTCTTTAGTTCGCCTCTTTGCAAGAAAGAAATGCGAATTCTTATGGTTGGTCTCGATGCTGCTGGTAAAACCACAATTCTGTACAAGCTCAAGTTGGGAGAAATTGTTACCACTATCCCAACCATTGGTTTCAATGTGGAGACTGTTGAATACAAAAACATCAGCTTTACTGTGTGGGATGTTGGTGGTCAGGACAAGATTAGACCTCTATGGAGGCACTATTTCCAGAACACACAGGGCCTCATCTTTGTGGTTGATAGCAATGACAGAGACCGTGTAGTTGAGGCAAGGGATGAGCTTCACAGGATGTTAAATGAGGATGAATTAAGAGAAGCTGTGTTGCTTGTTTTTGCGAACAAACAAGATCTTCCAAATGCAATGAATGCNNCTGAAATCACCGACAAGCTTGGCCTTCATTCTCTCAGACAACGACACTGGTATATCCAGAGTACATGTGCTACTTCTGGAGAAGGGCTATATGAGGGACTGGATTGGCTTTCAAACAACATCGCCAGCAAGGCCTAATGCAATGGTACTATGCTTCTTGTGTTGCTATATCCGGAGAAATAAACATCATTGTCTCGAGATTTTAAATATCTGTTCAGCTCACAATTCTGGGGAAGGCCTTACCCTTCTTCACTCTCTATGGTTTATGTCAAAGACCATGACATAGTTTACACATTGCTGGATGCACATTGGCAATGTAATGATATTTTAGTATAATATCTGGTTTTGAAACTTGGCGCAGCCGTGTGCACCATTTTGTTGTCCTGTGTGTCTGATGTTGCAATGGGTGTACAAAATGTAATACAGATCAATAGTAAGTATCGGA

Seq. 5 (SEQ ID NO.: 5): AC4: (no homology)

ACGGGACCTGGTCAATACTAATGTATCAGTCAACCAGCTCGAAAATCCACAAAATATAGAAGGGGAGGGAGGATCACCAAGGATAAACCATCTGAACCCAGACGACAACCTCCTTCTTCTTCTTCGATCCCTTAGGGAAGAGATACCCCGATCACCTGGATTAGGAAATAAGAGGAGCAAAATAACTTCAGAAACAGGAGGAATAAAGAGATCTAGTAAGGAGAGGGGAAGCACAAACTCTGAACCTTGGAAATGTGAAGCAGAGTAATGGTCTAACAGAGTTCACCATCGACTAGTGGAAGCACAAGCATAAGAACATCCAAAGGAGAAGGAGCTTAAGTCGGTGGTTCCAGCGACATG

Seq. 6 (SEQ ID NO.: 6): MOT Variant

GAATTCGCGGCCGCAAGAGAAAGAGAGCTGAGAAAGAATGGGTGAGAAGCCAGTATCTGGAGGTGTTTGGCCTACTGTTAAGCCATTTATTAATGGAGGTGTTTCTGGTATGCTTGCTACCTGTGTTATTCAGCCTATTGATATGATAAAGGTGAGGATACAATTGGGACAGGGATCAGCAGCTGATGTTACCAAAACCATGCTTAAAAATGAAGGCTTTGGTGCCTTTTACAAGGGTCTGTCAGCTGGGCTTCTTAGGCAGGCAACCTACACAACTGCCCGACTTGGGTCATTCAGAATTTTGACGAACAAGGCCATTGAGGCTAATGAAGGGAAGCCCTTACCTCTGTACCAAAAGGCTTTGTGTGGTCTAACTGCTGGAGCAATTGGTGCAACTGTTGGCAGTCCAGCAGATTTGGCCCTCATTCGTATGCAAGCTGATGCTACCTTGCCTTTAGCACAGAGACGCAATTACACAAATGCATTCCATGCACTCTCCCGTATTGCGGTTGATGAGGGAGTTCTAGCCCTCTGGAAAGGTGCTGGCCCAACAGTAGTAAGGGCAATGGCATTGAACATGGGTATGCTTGCCTCTTATGATCAGAGTGTGGAGTTCTTCAGGGACAACCTTGGCATGGGCGAGGCTGCTACAGTAGTAGGGGCCAGCAGTGTCTCTGGGTTCTTTGCTGCTGCTTGCAGTTTACCATTTGATTACGTCAAGACCCAGATTCAGAAAATGCAGCCAGATGCTGAAGGAAAATTGCCCTACACTGGTTCTTTCGATTGTGCCATGAAGACTTTGAAGGCAGGAGGACCCTTCAAATTTTACACTGGATTTCCAGTATATTGTATTAGGATTGCCCCTCATGTTATGATGACTTGGATTTTCCTTAACCAAATTCAGAAGGTGGAGAAGAAAATCGGATTGTGATTGTTGCAAAAAAAGATACATCCTCTCAAGTTGAGCTTTATTAGAAATAACATCTTCGCCTTGTTGTATTAGTACTGTTTTCGCTCTTTCTTTATCCTCACGCCTTCAAAGGCTTTAAGATTTTTGTGGTGATACATTGACTCGCGGAAATTTAGGGTTAGACATTTGGTCTTTTCAATATTCCTACCAATATAGTTTTGGGAAGATTACTTTATCCAAACTGATGGGAAGATTCTTTTAGCTGAATAATCTATGTACTTCAAAAACCGTCTTGAAGTAGGTAGTATGGAGTTCACCAATTTTGGTGTCATCTTGAACTTGATCTTGTTGCCTATTTTTGGATATACACTCATTTGTTAGCATCCTTCCTGGTATGAGCTATTGAGTATTATTGGAGTAAAAATGCATCCTAATGTTCTTGCTCCATTTGGATATATAGTTTTTTCATGCACCGCGGCCGCGAATTC

VIII. Identification of Promoter Regions 11. Isolation of Genomic Clones

11.1 A genomic library of Solanum tuberosum var. Solara in Zap-ExpressVectors (Stratagene) (750 000 Plaques were screened) was screened. cDNAfragments from the differential display were used as probes.

11.1.1. UBL (FIG. 23)

Three phage were isolated in the third screen, the in vivo preparationrevealed that two of them were identical and the third did not contain5, region of the UBL gene. One of the two identical phage was used for aPCR-approach. The clone was sequenced with an oligonucleotide (SEQ IDNO.: 18) (GCT TTC CGC CCA TGG ATT CC) reading into the promoter. Fromthis sequence, an additional oligonucleotide was deduced (a BamHI-siteadded) and used to make a PCR with the reverse-primer (Stratagene). Thefragment was cloned into pGemT (Promega) and sequenced. The cloning intothe pBI101 (Jefferson et al. 1987 EMBO 6) was made as BamHI fragment.Transgenic potato lines were generated containing the UBL promoter GUSconstruct as described previously. No detectable GUS activity wasobserved in a variety of tissue including stem and leaf. Tubers wereharvested and a number of transgenic lines were found to exhibit GUSexpression.

11.1.2. MOT (FIG. 24)

Six clones were isolated from the library. Two of them were to besequenced with a gene-specific primer (SEQ ID NO.: 19) (CCA GGA GAT GGGAAT GGA GAC CG), oligonucleotides were deduced for both clones. MOT6 andMOT3 fragments were isolated in combination with a universal Primer(Stratagene) and cloned into pGemT. MOT3 was cloned as BamHI fragment inpBI 101, MOT6 as BamHI/XbaI fragment.

12. Construction of Antisense/Sense Constructs

12.1.1. UBL-antisense Construct

A BamHI (internal restriction-site bp 301)/Asp718 (at 3-Prime end of thecDNA in the vector pBluescript) fragment was cloned into pBinAR. pBinARis a derivative of pBin19, containing a 35S-Promoter (Hoefgen undWillmitzer 1990, Plant Sci., 66,221-230).

12.1.2. MOT-antisense/sense Constructs

Oligonucleotides with restriction sites 5 prime BamHI base pairs 292-315of cDNA and 3 prime SalI base pairs 993-969 were used for PCR. Thfollowing fragments were cloned into pGemT:—BamHI/SalI-fragment inpBinAR (sense) and pBluescript (stratagene) and BamHI/Asp718 fragmentfrom pBluescript in pBinAR (antisense).

12.1.3.16-8 sense/antisense Constructs

Oligonucleotides with a restriction site 5 prime BamHI bp 6-29 cDNA and3 prime XbaI (sense) or Asp718 (antisense) bp 682-662 cDNA were used.Fragments were cloned into pGemT, and from there into pBinAR.

12.1.4. UBL-1, MOT6 and MOT6 Promoter Sequences

The sequences of these promoters are given in FIGS. 25, 26 and 27.

Other modifications of the present invention will be apparent to thoseskilled in the art without departing from the scope of the presentinvention.

39 1 423 DNA Tobacco FNR cDNA 1 tttttttttt tttttttttt tttttttttttttttttttt ttcccaaaaa atgaaattaa 60 aatttcaaag gaaaaattta cctatctacatggatgcagg gggagagaag cataaagttg 120 gctcatattt gtacaaagaa aagtaaaaatatttagtaga cttcaacatt ccattgctct 180 gccttcttca attgcttctt gtagtccgcccagacaatac catctctttc agcaagagca 240 gacataattt catcaattcc ctgctccatgcccttgagtc cacacatgta gatgaaggtg 300 ttgtcttttt ggagcaaagt ccatagttcttcagcatatt gagccattct ggtttgaatg 360 tacatctttt caccctttcc gttcgtttgctctctgctca cagcaaagtc caatctgaag 420 ttt 423 2 870 DNA ArtificialSequence Homology to ubiquitin carboxyl-terminal hydrolase from human,Drosophila and yeast 2 gggctgcagg aattcgaggc cgctagagag agttaaaatagaggaaagga atccatggcg 60 gaaagcacag gctctaagaa gagatggctt cctcttgaagctaaccccga tgtcatgaat 120 cagtttcttt ggggtcttgg tgttccaccg aatgaggccgagtgctgtga tgtttatggg 180 ttagatgaag aacttctgga gatggtgcca aagccagtgcttgctgtttt atttctctat 240 cctctcacat ctcagagtga agaagagaga ataaagcaagacagcgaaac aaaggtgcag 300 gatcccagta gtacagttta ctacatgaaa caaacagtgggaaatgcatg cggaacaatt 360 ggccttcttc atgctattgg gaatatcacc tctcagataaaacttaccga gggttcattc 420 ttggacaagt tctttaaatc aacctcaagc atggacccaatgcagcgtgc tttgttcctt 480 gaaaatgata gggaaatgga agttgctcat tcagtggcagccactgctgg tgatactgag 540 gctaccgacg atgtgaacgc tcatttcatc tgcttcacctgtgttgatgg acaactctat 600 gaacttgatg gaaggagggc tggacctatt acacatggcgcatcctctcc aaacagctta 660 ttaaaggatg cagccagagt tatcaaaaag ataatcgagaaaaatccaga ctcaatcaac 720 ttcaacgtta ttgctatttc ccaaaacgtt taggccaatctagaggcttt tatcgatgag 780 atggtttaaa ccaattttag cttttcatgt ttctgccgtttccagtacta tgtttcttct 840 tgtttgcaat aagttacttt tgagaaaaaa 870 3 712 DNAArtificial Sequence Homology to auxin-repressed protein from strawberry3 tgttctatcc cagcggacgc agaatttcct tttttattct tctcttcttc tcccctaaaa 60cgtgagccga ttggctaacc tgcaccatga gcttacttga caagctctgg gacgacaccg 120ttgccggtcc cctgccagat agtggcctcg ggaaactccg gaagtattct acttttagtc 180cgcgttcaaa ttccggcaag gaatcagaag tttccacacc gagatccttc accgaggaag 240caagtgagga cgtggtgaag gtgacgagaa gtatcatgat agtaaagcct tccgggagtc 300agaatagaga ttcacctcca gtttctccgg ccggtactac tcctccggta tctccttttg 360ccggttccgc tggaagagaa gcatttcggt tccggcggcg atcagcgtca tttgcatacg 420agaatgccag tggggttgga cccagaagcc ctcgtcctcc ttacgacctg tgagatatag 480tcgggttctc tttttttgtt atccctcttg aggcggttga atgtagtata gctagtcgac 540atactcaaca tgttcctggt tgagagtgtt gttttgtgtg gtgtttaatt tgtttgctta 600attttgtaaa tagtgcaagt ggttcttcat cttgcggatg ttgtgacgaa ggtttagcac 660aagatgtaag cgtccaagtt ggtcatgtat tctgctttgt attaaaaaaa aa 712 4 913 DNAArtificial Sequence ADP-RIBOSYLATION FACTOR 1 from potato belonging tothe family of GTP-binding proteins 4 tggacaatag agatctactg atttcatcctctctcatcgg ccgatcttcg attaacggag 60 atggggctgt ctttcactaa actctttagttcgcctcttt gcaagaaaga aatgcgaatt 120 cttatggttg gtctcgatgc tgctggtaaaaccacaattc tgtacaagct caagttggga 180 gaaattgtta ccactatccc aaccattggtttcaatgtgg agactgttga atacaaaaac 240 atcagcttta ctgtgtggga tgttggtggtcaggacaaga ttagacctct atggaggcac 300 tatttccaga acacacaggg cctcatctttgtggttgata gcaatgacag agaccgtgta 360 gttgaggcaa gggatgagct tcacaggatgttaaatgagg atgaattaag agaagctgtg 420 ttgcttgttt ttgcgaacaa acaagatcttccaaatgcaa tgaatgcnnc tgaaatcacc 480 gacaagcttg gccttcattc tctcagacaacgacactggt atatccagag tacatgtgct 540 acttctggag aagggctata tgagggactggattggcttt caaacaacat cgccagcaag 600 gcctaatgca atggtactat gcttcttgtgttgctatatc cggagaaata aacatcattg 660 tctcgagatt ttaaatatct gttcagctcacaattctggg gaaggcctta cccttcttca 720 ctctctatgg tttatgtcaa agaccatgacatagtttaca cattgctgga tgcacattgg 780 caatgtaatg atattttagt ataatatctggttttgaaac ttggcgcagc cgtgtgcacc 840 attttgttgt cctgtgtgtc tgatgttgcaatgggtgtac aaaatgtaat acagatcaat 900 agtaagtatc gga 913 5 360 DNAArtificial Sequence AC4, no homology. 5 acgggacctg gtcaatacta atgtatcagtcaaccagctc gaaaatccac aaaatataga 60 aggggaggga ggatcaccaa ggataaaccatctgaaccca gacgacaacc tccttcttct 120 tcttcgatcc cttagggaag agataccccgatcacctgga ttaggaaata agaggagcaa 180 aataacttca gaaacaggag gaataaagagatctagtaag gagaggggaa gcacaaactc 240 tgaaccttgg aaatgtgaag cagagtaatggtctaacaga gttcaccatc gactagtgga 300 agcacaagca taagaacatc caaaggagaaggagcttaag tcggtggttc cagcgacatg 360 6 1398 DNA Artificial Sequence MOTVariant. 6 gaattcgcgg ccgcaagaga aagagagctg agaaagaatg ggtgagaagccagtatctgg 60 aggtgtttgg cctactgtta agccatttat taatggaggt gtttctggtatgcttgctac 120 ctgtgttatt cagcctattg atatgataaa ggtgaggata caattgggacagggatcagc 180 agctgatgtt accaaaacca tgcttaaaaa tgaaggcttt ggtgccttttacaagggtct 240 gtcagctggg cttcttaggc aggcaaccta cacaactgcc cgacttgggtcattcagaat 300 tttgacgaac aaggccattg aggctaatga agggaagccc ttacctctgtaccaaaaggc 360 tttgtgtggt ctaactgctg gagcaattgg tgcaactgtt ggcagtccagcagatttggc 420 cctcattcgt atgcaagctg atgctacctt gcctttagca cagagacgcaattacacaaa 480 tgcattccat gcactctccc gtattgcggt tgatgaggga gttctagccctctggaaagg 540 tgctggccca acagtagtaa gggcaatggc attgaacatg ggtatgcttgcctcttatga 600 tcagagtgtg gagttcttca gggacaacct tggcatgggc gaggctgctacagtagtagg 660 ggccagcagt gtctctgggt tctttgctgc tgcttgcagt ttaccatttgattacgtcaa 720 gacccagatt cagaaaatgc agccagatgc tgaaggaaaa ttgccctacactggttcttt 780 cgattgtgcc atgaagactt tgaaggcagg aggacccttc aaattttacactggatttcc 840 agtatattgt attaggattg cccctcatgt tatgatgact tggattttccttaaccaaat 900 tcagaaggtg gagaagaaaa tcggattgtg attgttgcaa aaaaagatacatcctctcaa 960 gttgagcttt attagaaata acatcttcgc cttgttgtat tagtactgttttcgctcttt 1020 ctttatcctc acgccttcaa aggctttaag atttttgtgg tgatacattgactcgcggaa 1080 atttagggtt agacatttgg tcttttcaat attcctacca atatagttttgggaagatta 1140 ctttatccaa actgatggga agattctttt agctgaataa tctatgtacttcaaaaaccg 1200 tcttgaagta ggtagtatgg agttcaccaa ttttggtgtc atcttgaacttgatcttgtt 1260 gcctattttt ggatatacac tcatttgtta gcatccttcc tggtatgagctattgagtat 1320 tattggagta aaaatgcatc ctaatgttct tgctccattt ggatatatagttttttcatg 1380 caccgcggcc gcgaattc 1398 7 34 DNA Artificial Sequence5′-rolC primer. 7 8 29 DNA Artificial Sequence 3′-rolC primer. 8cccatggtac cccataactc gaagcatcc 29 9 45 DNA Artificial Sequence 5′-Suc2d oligonucleotides. 9 gagctgcaga tggcaaacga aactagcgat agacctttgg tcaca45 10 42 DNA Artificial Sequence 3′-Suc2 d oligonucleotides. 10gagactagtt tataacctct attttacttc ccttacttgg aa 42 11 36 DNA ArtificialSequence Oligonucleotide synthesized with BamHI and Asp718 restrictionsites. 11 ttggtaccaa ttgtgagcgc tcacaattgg atcctt 36 12 36 DNAArtificial Sequence Oligonucleotide synthesized with BamHI and Asp718restriction sites. 12 aaggatccaa ttgtgagcgc tcacaattgg taccaa 36 13 28DNA Artificial Sequence 5′-ANT primer. 13 aacggatcca tggcagatat gaaccagc28 14 29 DNA Artificial Sequence 3′-ANT primer. 14 ttggatcctt acaacacacccgcccaggc 29 15 10 DNA Artificial Sequence Tuber-specific cDNAfragments. 15 aagcgacctg 10 16 10 DNA Artificial Sequence Tuber-specificcDNA fragments. 16 gttggtggct 10 17 10 DNA Artificial SequenceTuber-specific cDNA fragments. 17 acgggacctg 10 18 20 DNA ArtificialSequence Oligonucleotide. 18 gctttccgcc catggattcc 20 19 23 DNAArtificial Sequence Gene specific primer. 19 ccaggagatg ggaatggaga ccg23 20 42 DNA Artificial Sequence Plasmid pBIN-IN8. 20 aaggttcattcccttcattt tatccttaat atttgatcag ag 42 21 30 DNA Artificial SequenceBamHI. 21 ggatccccca tcgaattcct gcagatggca 30 22 24 DNA ArtificialSequence BamHI. 22 tagaggttat aaactagagg atcc 24 23 20 DNA ArtificialSequence Oligonucleotide. 23 caggaaacag ctatgaccat 20 24 30 DNAArtificial Sequence Oligonucleotide. 24 tctagaaagc ttgtaaaacg acggccagtg30 25 32 DNA Artificial Sequence BamHI oligonucleotide. 25 atggatccggagaaacccca atttcagctc cg 32 26 32 DNA Artificial Sequence SaIIoligonucleotide. 26 atgtcgaccg gctcgaccaa catgttcata ac 32 27 1351 DNAArtificial Sequence DNA sequence encoding MOT isolated from potato. 27cgg cgt ccc aca ctt cgc atc tat agc ttt cgg tct cca ttc cca tct 48 ArgArg Pro Thr Leu Arg Ile Tyr Ser Phe Arg Ser Pro Phe Pro Ser 1 5 10 15cct ggt ttc cag tga gat gaa ctc taa ttc caa ttg ggc tta aac ctt 96 ProGly Phe Gln Asp Glu Leu Phe Gln Leu Gly Leu Asn Leu 20 25 30 tga ttc attcta ttt ttt ttt ttc tat ttt ttc cat tac cta att cat 144 Phe Ile Leu PhePhe Phe Phe Tyr Phe Phe His Tyr Leu Ile His 35 40 45 att cat tct ttt tttaaa aaa agc ttt cgt ctc gat tca ttt ggt ata 192 Ile His Ser Phe Phe LysLys Ser Phe Arg Leu Asp Ser Phe Gly Ile 50 55 60 atg ggt gtt aag gga tttgtt gaa gga ggt att gct tcg att att gct 240 Met Gly Val Lys Gly Phe ValGlu Gly Gly Ile Ala Ser Ile Ile Ala 65 70 75 ggt tgt agt act cac cca cttgat tta atc aaa gtc cgt atg cag ctt 288 Gly Cys Ser Thr His Pro Leu AspLeu Ile Lys Val Arg Met Gln Leu 80 85 90 cag gga gaa acc cca att tca gctccg gcg act gtt cac aat ctc cgt 336 Gln Gly Glu Thr Pro Ile Ser Ala ProAla Thr Val His Asn Leu Arg 95 100 105 cca gca ctt gct ttt cac act ggtgct gct aat cat act ttt tcc att 384 Pro Ala Leu Ala Phe His Thr Gly AlaAla Asn His Thr Phe Ser Ile 110 115 120 125 ccg gcg ccg tcg gtg gtt gctcca ccg cgt gta gga ccg gtt tct gta 432 Pro Ala Pro Ser Val Val Ala ProPro Arg Val Gly Pro Val Ser Val 130 135 140 ggt gtt aag att att caa caagaa gga gtt gct gct ttg ttc tcc ggt 480 Gly Val Lys Ile Ile Gln Gln GluGly Val Ala Ala Leu Phe Ser Gly 145 150 155 gta tca gct act gtt ctc cggaca gac act tta ctc tac aac cag aat 528 Val Ser Ala Thr Val Leu Arg ThrAsp Thr Leu Leu Tyr Asn Gln Asn 160 165 170 ggg ttt ata cga tat gct gaagca aaa atg gac cga tcc aga tac tac 576 Gly Phe Ile Arg Tyr Ala Glu AlaLys Met Asp Arg Ser Arg Tyr Tyr 175 180 185 atc atg cct ttg tcg aag aagatc gtt gcc gga tta atc gcc ggc ggg 624 Ile Met Pro Leu Ser Lys Lys IleVal Ala Gly Leu Ile Ala Gly Gly 190 195 200 205 atc gga gct gcc gtc ggtaat ccc gcc gat gta gcg atg gtc cgc atg 672 Ile Gly Ala Ala Val Gly AsnPro Ala Asp Val Ala Met Val Arg Met 210 215 220 caa gct gac ggc cgg cttccg atc tct caa cgc cgc aac tac aaa agc 720 Gln Ala Asp Gly Arg Leu ProIle Ser Gln Arg Arg Asn Tyr Lys Ser 225 230 235 gtg atc gat gca att tctcag atg agt aaa agc gaa ggg gta act agc 768 Val Ile Asp Ala Ile Ser GlnMet Ser Lys Ser Glu Gly Val Thr Ser 240 245 250 ctg tgg cgc ggt tca tctctt act gtg aac cgc gcc atg cta gtt acc 816 Leu Trp Arg Gly Ser Ser LeuThr Val Asn Arg Ala Met Leu Val Thr 255 260 265 gca tcg cag cta gca tcgtac gat cag ttc aaa gag act atc ctc gag 864 Ala Ser Gln Leu Ala Ser TyrAsp Gln Phe Lys Glu Thr Ile Leu Glu 270 275 280 285 aag ggg tta atg aaggat ggg ctt ggg aca cat gtg act tcg agt ttt 912 Lys Gly Leu Met Lys AspGly Leu Gly Thr His Val Thr Ser Ser Phe 290 295 300 gct gct ggg ttt gtggcg gcg gtg gca tcg aat cca gtg gat gtg att 960 Ala Ala Gly Phe Val AlaAla Val Ala Ser Asn Pro Val Asp Val Ile 305 310 315 aag aca cgt gtt atgaac atg aag gtc gag ccg gaa atg gcc cca ccg 1008 Lys Thr Arg Val Met AsnMet Lys Val Glu Pro Glu Met Ala Pro Pro 320 325 330 tat aat ggg gcc attgat tgt gca atg aaa act atc aaa gct gag ggg 1056 Tyr Asn Gly Ala Ile AspCys Ala Met Lys Thr Ile Lys Ala Glu Gly 335 340 345 cca atg gca ttg tataag gga ttt att cct aca atc tca agg caa ggt 1104 Pro Met Ala Leu Tyr LysGly Phe Ile Pro Thr Ile Ser Arg Gln Gly 350 355 360 365 cca ttt act gtggtg ctc ttt gtc aca ctg gaa caa gtc agg aaa atg 1152 Pro Phe Thr Val ValLeu Phe Val Thr Leu Glu Gln Val Arg Lys Met 370 375 380 ctc aag gat ttttaa tga tga tga cga aga aaa aaa aaa tta atg gga 1200 Leu Lys Asp Phe ArgArg Lys Lys Lys Leu Met Gly 385 390 ttt tag tat taa gaa ttt aaa aaa aagtta agt tta att tat gtt ttt 1248 Phe Tyr Glu Phe Lys Lys Lys Leu Ser LeuIle Tyr Val Phe 395 400 405 aag ttt tta agt ttg gga aaa gtg ata cta tgttgt gtt cta ata tta 1296 Lys Phe Leu Ser Leu Gly Lys Val Ile Leu Cys CysVal Leu Ile Leu 410 415 420 tta tta ttg tta ctt cta tat gaa aaa tga gttctt gtt tgg tgg aaa 1344 Leu Leu Leu Leu Leu Leu Tyr Glu Lys Val Leu ValTrp Trp Lys 425 430 435 aaa aaa a 1351 Lys Lys 440 28 20 PRT ArtificialSequence Amino acid sequence encoding MOT isolated from potato. 28 ArgArg Pro Thr Leu Arg Ile Tyr Ser Phe Arg Ser Pro Phe Pro Ser 1 5 10 15Pro Gly Phe Gln 20 29 7 PRT Artificial Sequence Amino acid sequenceencoding MOT isolated from potato. 29 Phe Gln Leu Gly Leu Asn Leu 1 5 30355 PRT Artificial Sequence Amino acid sequence encoding MOT isolatedfrom potato. 30 Phe Ile Leu Phe Phe Phe Phe Tyr Phe Phe His Tyr Leu IleHis Ile 1 5 10 15 His Ser Phe Phe Lys Lys Ser Phe Arg Leu Asp Ser PheGly Ile Met 20 25 30 Gly Val Lys Gly Phe Val Glu Gly Gly Ile Ala Ser IleIle Ala Gly 35 40 45 Cys Ser Thr His Pro Leu Asp Leu Ile Lys Val Arg MetGln Leu Gln 50 55 60 Gly Glu Thr Pro Ile Ser Ala Pro Ala Thr Val His AsnLeu Arg Pro 65 70 75 80 Ala Leu Ala Phe His Thr Gly Ala Ala Asn His ThrPhe Ser Ile Pro 85 90 95 Ala Pro Ser Val Val Ala Pro Pro Arg Val Gly ProVal Ser Val Gly 100 105 110 Val Lys Ile Ile Gln Gln Glu Gly Val Ala AlaLeu Phe Ser Gly Val 115 120 125 Ser Ala Thr Val Leu Arg Thr Asp Thr LeuLeu Tyr Asn Gln Asn Gly 130 135 140 Phe Ile Arg Tyr Ala Glu Ala Lys MetAsp Arg Ser Arg Tyr Tyr Ile 145 150 155 160 Met Pro Leu Ser Lys Lys IleVal Ala Gly Leu Ile Ala Gly Gly Ile 165 170 175 Gly Ala Ala Val Gly AsnPro Ala Asp Val Ala Met Val Arg Met Gln 180 185 190 Ala Asp Gly Arg LeuPro Ile Ser Gln Arg Arg Asn Tyr Lys Ser Val 195 200 205 Ile Asp Ala IleSer Gln Met Ser Lys Ser Glu Gly Val Thr Ser Leu 210 215 220 Trp Arg GlySer Ser Leu Thr Val Asn Arg Ala Met Leu Val Thr Ala 225 230 235 240 SerGln Leu Ala Ser Tyr Asp Gln Phe Lys Glu Thr Ile Leu Glu Lys 245 250 255Gly Leu Met Lys Asp Gly Leu Gly Thr His Val Thr Ser Ser Phe Ala 260 265270 Ala Gly Phe Val Ala Ala Val Ala Ser Asn Pro Val Asp Val Ile Lys 275280 285 Thr Arg Val Met Asn Met Lys Val Glu Pro Glu Met Ala Pro Pro Tyr290 295 300 Asn Gly Ala Ile Asp Cys Ala Met Lys Thr Ile Lys Ala Glu GlyPro 305 310 315 320 Met Ala Leu Tyr Lys Gly Phe Ile Pro Thr Ile Ser ArgGln Gly Pro 325 330 335 Phe Thr Val Val Leu Phe Val Thr Leu Glu Gln ValArg Lys Met Leu 340 345 350 Lys Asp Phe 355 31 9 PRT Artificial SequenceAmino acid sequence encoding MOT isolated from potato. 31 Arg Arg LysLys Lys Leu Met Gly Phe 1 5 32 37 PRT Artificial Sequence Amino acidsequence encoding MOT isolated from potato. 32 Glu Phe Lys Lys Lys LeuSer Leu Ile Tyr Val Phe Lys Phe Leu Ser 1 5 10 15 Leu Gly Lys Val IleLeu Cys Cys Val Leu Ile Leu Leu Leu Leu Leu 20 25 30 Leu Leu Tyr Glu Lys35 33 8 PRT Artificial Sequence Amino acid sequence encoding MOTisolated from potato. 33 Val Leu Val Trp Trp Lys Lys Lys 1 5 34 300 PRTArtificial Sequence MOT potato protein. 34 Leu Ile Lys Val Arg Met GlnLeu Gln Gly Glu Thr Pro Ile Ser Ala 1 5 10 15 Pro Ala Thr Val His AsnLeu Arg Pro Ala Leu Ala Phe His Thr Gly 20 25 30 Ala Ala Asn His Thr PheSer Ile Pro Ala Pro Ser Val Val Ala Pro 35 40 45 Pro Arg Val Gly Pro ValSer Val Gly Val Lys Ile Ile Gln Gln Glu 50 55 60 Gly Val Ala Ala Leu PheSer Gly Val Ser Ala Thr Val Leu Arg Thr 65 70 75 80 Asp Thr Leu Leu TyrAsn Gln Asn Gly Phe Ile Arg Tyr Ala Glu Ala 85 90 95 Lys Met Asp Arg SerArg Tyr Tyr Ile Met Pro Leu Ser Lys Lys Ile 100 105 110 Val Ala Gly LeuIle Ala Gly Gly Ile Gly Ala Ala Val Gly Asn Pro 115 120 125 Ala Asp ValAla Met Val Arg Met Gln Ala Asp Gly Arg Leu Pro Ile 130 135 140 Ser GlnArg Arg Asn Tyr Lys Ser Val Ile Asp Ala Ile Ser Gln Met 145 150 155 160Ser Lys Ser Glu Gly Val Thr Ser Leu Trp Arg Gly Ser Ser Leu Thr 165 170175 Val Asn Arg Ala Met Leu Val Thr Ala Ser Gln Leu Ala Ser Tyr Asp 180185 190 Gln Phe Lys Glu Thr Ile Leu Glu Lys Gly Leu Met Lys Asp Gly Leu195 200 205 Gly Thr His Val Thr Ser Ser Phe Ala Ala Gly Phe Val Ala AlaVal 210 215 220 Ala Ser Asn Pro Val Asp Val Ile Lys Thr Arg Val Met AsnMet Xaa 225 230 235 240 Val Glu Pro Glu Met Ala Pro Pro Tyr Asn Gly AlaIle Asp Cys Ala 245 250 255 Met Lys Thr Ile Lys Ala Glu Gly Pro Met AlaLeu Tyr Lys Gly Phe 260 265 270 Ile Pro Thr Ile Ser Arg Gln Gly Pro PheThr Val Val Leu Phe Val 275 280 285 Thr Leu Glu Gln Val Arg Lys Met LeuLys Asp Phe 290 295 300 35 299 PRT Panicum miliaceum 35 Met Ala Asp AlaLys Gln Gln Gln Ala Val Ala Pro Ser Ala Ala Trp 1 5 10 15 Met Met ValLys Pro Phe Val Asn Gly Gly Ala Ser Gly Met Leu Ala 20 25 30 Thr Cys ValIle Gln Pro Ile Asp Met Val Lys Val Lys Ile Gln Leu 35 40 45 Gly Glu GlySer Ala Ala Thr Val Thr Lys Lys Met Leu Ala Asn Glu 50 55 60 Gly Ile GlySer Phe Tyr Lys Gly Leu Ser Ala Gly Leu Leu Arg Ala 65 70 75 80 Thr TyrThr Thr Ala Arg Leu Gly Ser Phe Arg Val Leu Thr Asn Lys 85 90 95 Ala ValGlu Ala Asn Glu Gly Lys Pro Leu Pro Leu Leu Gln Lys Ala 100 105 110 ValIle Gly Leu Thr Ala Gly Ala Ile Gly Ala Ser Val Gly Ser Pro 115 120 125Ala Asp Leu Ala Leu Ile Arg Met Gln Ala Asp Ser Thr Leu Pro Ala 130 135140 Ala Gln Arg Arg Asn Tyr Lys Asn Ala Phe His Ala Leu Tyr Arg Ile 145150 155 160 Val Ala Asp Glu Gly Val Leu Ala Leu Trp Lys Gly Ala Gly ProThr 165 170 175 Val Val Arg Ala Met Ser Leu Asn Met Gly Met Leu Ala SerTyr Asp 180 185 190 Gln Ser Val Glu Leu Phe Arg Asp Lys Leu Gly Ala GlyGlu Leu Ser 195 200 205 Thr Met Leu Gly Ala Ser Ala Val Ser Gly Phe CysAla Ser Ala Cys 210 215 220 Ser Leu Pro Phe Asp Tyr Val Lys Thr Gln IleGln Lys Met Gln Pro 225 230 235 240 Asp Ala Asn Gly Lys Tyr Pro Tyr ThrGly Ser Leu Asp Cys Val Met 245 250 255 Lys Thr Leu Lys Ser Gly Gly ProPhe Lys Phe Tyr Thr Gly Phe Pro 260 265 270 Val Tyr Cys Val Arg Ile GlyPro His Val Met Leu Thr Trp Ile Phe 275 280 285 Leu Asn Gln Ile Gln LysPhe Glu Lys Asp Met 290 295 36 37 DNA Artificial Sequence PCR fragment.36 atggatcccg cttctcctct ttatatatag ttatggg 37 37 3933 DNA ArtificialSequence UBL-1 promoter nucleic acid sequence. 37 ggatcctttc acctcctaatacaaaagttt ccattttttt aagcaggcaa tgatagtttt 60 tgttaatgct aatctttgttaccatatatg gtctttatca gagcctccag acatccggaa 120 ctggttctct agctatgtttatgaatctcc aaaagtggat actattcaag attccatact 180 tccagatcat gagaaagaattagatgacaa agtgtgtacg aatggataca gtggcggtga 240 ggaacctcag aattttaggaattcattagg aactcctttt atccatgatg acaagtatga 300 gcatcaaact gcctcaaaggtaaacttaga accttcagct gcatcaaact cctttcatac 360 atcctcttga tctacatcaattctttgtga actcatgctt tagatgttga tttattgaat 420 gtacactcaa agtaaacgtagaaccttaag ctgtagatca acaaacagat ggattttatc 480 tttctgcgat tgtacgaacctttttggctg gagggatcag tacctagaat atacaataag 540 attacattga gttacagtgttggatcacat agttgaacat atgtacaaaa caaagacaga 600 aagaaacatt aaaagatctattctgtcttt agttagttag aaacttaggt attttcagtt 660 ggctggtagc tatgcatataaaccatctca tctccgattc tgttagttat acaattgttt 720 ctaccatgca aagaagataactgacacttc agctacataa ttgaggtcta ccttaccata 780 ttgtcataag ttcccttgatgatttccttt gtgtttgtat gcttcgaagg atcaggaggc 840 tgatgggaca aagaacgcaagaatatccaa tgaaatgtct catgagagaa tttctcaaca 900 gacactaaat cacaagacaacggagaacac caattgtggt tcaccaagat acattgacat 960 ggtcttcaaa gagagtgatggagaacactt ggagaccatt tttcctcaag aagttaactg 1020 caaagtatcc tgcaccatcaatcattctag ctgtgaaggt gaaaaattat acagacatcc 1080 aattcacagg aaggattctgcagagaacag ttcgaaatct aaagatagtg ttgaacctgc 1140 tgatgatgtg caatctaaaaataggatgga gatgagtgtg ttaagtcaga agttatccaa 1200 acggaaagca gcagaaatcatcgacaaaga aaatcacata aatgactttg gagagaatgg 1260 ttttatatca accagaaagagtagaaatag tcaagtgcag aacaaaagtc ctttgccaac 1320 gccagctgca gttcagtctcctttaagtgg agtcactgtt gcatcaaact gccacaagca 1380 gggtttgact agaaaggtactcacagaaac aaccaacttg catcctagtg ctttggaaaa 1440 aacaggaaaa tggcggtgcccccaaaggac taagccaaat attggtcctc ctctaaagca 1500 gcttcgacta gagcaatgggttcgtcgagc ttaagtctaa tacattctta tgaagagaaa 1560 atggatatca agaatggtagaattcaaaag aggtttgtgc atgttagcta gtgaaagatg 1620 tgagaacaag acttggcacaatgctagagt tactatatcg tggttgtcaa tttacaatgc 1680 aaatgagatc tattaaattgacaaccacga tatttagatt ttttttaata ggtttggcct 1740 tgagtctaat tttgttggacattcacatga tcaaatagat tgaagtattt tttaattagg 1800 agcgtttcaa ctcacttattaggtctattc gacacaagtt tagattgatt atcttcactt 1860 gtttcggaca ccaagttattaaactgaaaa atataaaggc gaaatggtct ggtggaccct 1920 catacttgta tgtgtttgttttgtgaatcc ttctacttgt ttctttgtca tctgaaccct 1980 tgaactcatc aaaacacaatattttaaaca cgttttttac tactcaaatg tgtgtgtatt 2040 acaaatgcct gacacgtaattttaaaaata attataaaat gacacgtata attataaaat 2100 gacacatgta tatatgatcactccatgtca tttttccttt tattatattt gattaatata 2160 cctacacttt ctctttccaatttttatttc tctttgtcat agccatctgc atctgcattt 2220 ccattcccac aatctcttttcaactttttc ttatcttcct tacccttttc ctttttccaa 2280 tctcttctac tctttccattcaagtaaaaa atgttggtac gatttctgat tgttcacaat 2340 ctcgtcgaag ttcggagttgattttgggtt ctgttggatt gggattttgg tggtgaccgg 2400 tggcgctaag gaaaagtgggggtctattgg gtggtctgat tgtttctagt tgttcacaac 2460 tagcggcgtt gtgaaagtcgagacttattg gcagagttag tttggtgttt ttgtcttagt 2520 tgttgttgtt gttggttctcatttatagtt gttggttgaa gcttgtcgga gatggtgaga 2580 acgaaggcaa tttggtcggagaaggtgagg aggacgagaa gaaggcaata agtttgagtt 2640 tggtttggaa ctgaaacaaggggtcaatgt caaatatatt tgagtttttt tgtttgattt 2700 tcaacttatc taggtaggttttccaattta ttttcgaatt tatttgttgt ttagtttgga 2760 tttgatctct atttgtgtctttgtttgatt ttggggtttt agtcgattac attgattttg 2820 ttgatttttt ttggggattttgtatttttg tgtctaatgt tagtgtaaga tattttggta 2880 gtagtgttat gccttccatggcatttttca taaaaaaaaa agaagagttg tataataaca 2940 agaaacgata gagaagaaatttggggggaa aagatcaaaa aagaggccaa gaaaagctca 3000 aattttgtcc aacaatggtgttagatgcaa ataggaagat gatggcttta caaagcccta 3060 atgtcactgt taaacccttttccaagggtc tcacgctcct aataggtgtg tgtcacacac 3120 tctttgatat tcactgccacataggatgtc aagtcaaaaa tagtgtttaa aatattgtgt 3180 tttgataagt tcaggggttcagatgacaaa tgggcaagta gaagggttca caatacaaac 3240 atatacaagt ataagagtccaccagaccat ttcsccaaat ataaatagat gggaagtgta 3300 gatctcctca aaattcttttagtaacagta aacactcata tacaaaaata tgtaatatga 3360 agttatgtgc aaccaaataaaattttaaaa attgaaactt tctttttttg tttcccaccc 3420 tatatatcag ggaccacattggagtctgga ttaaatttga atcgtgtatt gtagggatca 3480 ttttccaata gaattttctccatactccgg aaaagttgaa aacttgctct tttgataaaa 3540 atgttgttta aaagggaaatatatttgaaa caatcaaatg tgttcctgga aaagtatctc 3600 gtgttaataa cttgctaaatatttagcatt ctaatatacc tttgaattta aattcttcat 3660 cttgtggttt ttttcaactttaaatattca aaatactggt aaattgattt agtgatctat 3720 tacaatttta gttttaggtccaatcaaatc tctccaacat tttatttttt attcttaaaa 3780 tatttctttt ataaaattatattttattta aattgtaaaa acaaacaaac aaaaaatgat 3840 aaagaaaaat aagaagacgagggtgctaga aaatgataaa aaccccccca ccataaagcc 3900 cttcccataa ctatatataaagaggagaag gcg 3933 38 1031 DNA Artificial Sequence MOT3 promoternucleic acid sequence. 38 ggatccatta gttacacatt gtagactttt aacttttcaatggcataatt cctcacgtaa 60 tcaaataaat aattttttct cttttctatc taacattttctcttgaaaaa tataaagtag 120 tggtaactat tgtccaattg taattcaaat atgaggcatcttttcattat acaatcgact 180 tgaagtagaa tatttataag attttatgcc ttattgagaatctaattgtt ataaatagtt 240 tataaaagtc aatttctttt aaatttatta ttcgtatcagttaaaaaaat tatatcccaa 300 cattgttatt cgtattgtta gtaaaaatta actgcatgtctggcttttct tgaacatagt 360 tgatgatcta ttgatgcgcg atcttcattc atttgttgatctaattatgc gtataaatta 420 taatcaaata aaacgacatg tttaagtggt taatttgtctacgtaacaaa aaattgagta 480 ttcatacaaa aacttaacaa aaattgaatc aaaattatctaatataaaca tttatatatt 540 caatcagaac ataccatact tcaaatatct aaatagctaaaaaataataa tacaaatgaa 600 gtgaccggat caagattttt gagttatatt acacttttcatttatggctg agtcaaaatt 660 ttcactaaaa aattcaaaat taacacgcaa taaaacaaaacaaaattcaa cacctaaaaa 720 gagtcaaatg aatgaaaaat cccctcgatc ctacttaactccgcccccaa cttccaactt 780 cattattaca accaaaaaat atttccattg accaaaggctcctactttcc ttccgccgca 840 gagaaaagta tactgaaaga acccgcgttg tatacaaaacctaatttccc tttcctttcc 900 tttccctttc ccttttttcc cttataaatt cgtttcttcctcttccttct caactcacaa 960 ttttatgtct cacagactca acgttccaca cttcgcatctatagctttcg gtctccattc 1020 ccatctcctg g 1031 39 2813 DNA ArtificialSequence MOT6 promoter nucleic acid sequence. 39 ggatctcatt ttctaaacatgcttgaaatt tatggtctaa aataagtcac agatgattat 60 gcggctatat aacaatatttgcttgaactc cattttcgaa cttatcatcc ggagttaggt 120 gagtctaatt tgttacttcggatctttgat agatatgaac tatcctatta ggcgtggcac 180 aagtccatgt ttggtttgggtaccgctatg ttggacttga ttgaattttg atcgttggat 240 atcgcttgat gatatattccaatgtttaaa ttgaattttg attcatatga atttttaaaa 300 tcatcaaaca atacatgacaaagaacaagt tcatatgcta catagatgtg tttgggctta 360 attgacatag attaaagaataaatttataa tgcattgagt tcaatgagct tagtaataaa 420 tgtatgcaca aagccaattgtataaaaatg tgcaaattac tcaaccaaat ctaaaaataa 480 gacgacttta gactaattttataacatctt aattgaccaa gtcgacatga ttttatttca 540 aaccacatat atatgctctcttttttttag aaagaaaaaa taaacaaatt tacaccccaa 600 agttttactt gtgggataaagtagctttgg actttcaaaa ttgttgttat aaccagataa 660 atgctgattt tcgtttttcaattttgtctt tataaagaaa tgaatttgga ttctaactca 720 atcataaaaa ttagttaagagatgggaata ttgtctaaac catattaaag agatccccac 780 ccccacccac cgactcgaaagcaagaggca agagcgcaac aactacatga aagccttatg 840 agtaaggtta atcgaagtcagaaaaagttt attggcaaga gggaatcaaa tattttaaaa 900 tatttgggtc ctccactcatcaaaatttat atgatatttt ttccttttra gttcgtttta 960 aaaagaacag aatcttctatatttagtaac aacttaactt taactrcaca tattttaggt 1020 aagtaaattt catatttttaccattaataa gatgatttat agccgcatag atatctatga 1080 cttattttaa gctataaattttaaaaatct ttcttttatt cttaaacttc atgccgaagc 1140 gaacacctaa agaataatagtattttattt aatcacaaag aacaagtaac accatgttac 1200 gttaatatag gaacaatattatatcatgcc cacctccaaa ggacaacaaa aaaagaaaga 1260 aaaaaaaaag tcaaaatggcttcttagcca ccaaaaaaaa gttttattta attaaaagct 1320 cttttttaat ttcacacgtttaagggagaa taattctaag tagagtactt tgacctaaga 1380 aattttgaaa aagtcatagtcaaaactata aaagtcaaaa agaattgaat ccattttcac 1440 ataattttca atatcacatttagtaatgat tgataaattc agtactaaaa taaatcaaaa 1500 attcataaat ttaagtttgactttgcttct ctttaataaa ataatttaaa tggtatgaaa 1560 tcatattaat cagatcgataaatttagaat agtaaataca taaacaaaag gttttattta 1620 tgggatcata agttgttgcctagtaggtaa aggagcgtgt gctaggcaca tgcataaggg 1680 tcctacaact tctactactagtgagcccat ataagtgaaa ctcgaagatt gttctcattt 1740 aataatatct tattcttcgtttatattatt atttgtattt tttttcttcg attatcgtat 1800 tatatatatt gctcactatgttcagcataa ctgcttcatt gttgtatttc ccttttcata 1860 cttgatttta ttattctttaagccgagagt ctattgtcca attgtaattt aaatatgagg 1920 cctcttttga ttatacaattgacattttaa gtagaatatg ttttgagaat ctaattgtta 1980 taaatagcgt ataaaagtcaatttctttta agttcattat ttgtgtcagt aaaaaaaaaa 2040 aactatattt caaaattgttattcgtactg ttgttagtaa aaaataactg catgtctggc 2100 ttttcttgaa cggtctattgatgcgcgatc ttcatccatt tgctgatcta attatgcgta 2160 taaattataa taaaaataaaacgacatatt ttaagtggtt aatttgtcta cgtaacaagc 2220 aattgagtat tcatacaaaaacttaacaaa atttgaatta aaattatcta atataagcat 2280 ttatatcata tatttaagtattcaatcaga gcataccata tttcaaatat ctaaatagct 2340 aaaaaaaaat acaaatgaagtgactgggtc aagatttttg tgttatatta cattttccat 2400 gtgtggacgt ctgagtcaaaattttcacta aacaatcaca aaacacaaaa caaaattcaa 2460 cacctaaaaa gagtcaaataaatgaaaaat cccctcgatc cgacttaact cccccccgac 2520 ttctaacttc attattacaaccaaaaaata tttccattga ccaaaggccc ccactttcct 2580 tccgccgcag agaaaagtatactgaaagaa cccgcgttgt atacaaaacc taatttccct 2640 ttcctttcct ttcccttccctttttccctt ataaattcgc ttcttcctct tccttctcaa 2700 ctcacaattt atatgtctcacagactcaac gttccacact tcgcatctat agctttcggt 2760 ctccattccc atctcctggtttccagtgag atgaactcta attccaattg ggc 2813

What is claimed is:
 1. A method for the selective induction orsuppression of sprouting in a vegetative storage organ of a plantcomprising incorporating into the genome of said plant by transformationa DNA construct comprising a first polynucleotide sequence comprising atleast one DNA sequence encoding an invertase or an inorganicpyrophosphatase operably linked to a tissue or organ selective promoterregion and optionally to a transcription terminator region and a secondpolynucleotide sequence comprising at least one DNA sequence operablylinked to and controlled by a controllable promoter region andoptionally to a transcription terminator region whereby the DNAsequence(s) in said first polynucleotide sequence is expressed duringdormancy of the vegetative organ derived from said transgenic plantresulting in effective suppression of sprouting and the said suppressionis neutralised by inducing expression of the DNA sequence(s) in saidsecond polynucleotide sequence from said controllable promoter region byexternal application of an inducing substance thus making restoration ofsprouting of said vegetative storage organ dependent on the applicationof the inducer, so that sprouting is selectively induced or suppressed.2. A method according to claim 1 for the selective induction orsuppression of sprouting in potatoes comprising forming a transgenicpotato by incorporating into the genome of a potato by transformation aDNA construct comprising a first polynucleotide sequence comprising atleast one DNA sequence encoding an invertase or an inorganicpyrophosphatase operably linked to a tissue or organ selective promoterregion and optionally to a transcription terminator region and a secondpolynucleotide sequence comprising at least one DNA sequence operablylinked to and controlled by a controllable promoter region andoptionally to a transcription terminator region whereby the DNAsequence(s) in said first polynucleotide sequence is expressed duringdormancy of the tuber derived from said transgenic potato resulting ineffective suppression of sprouting and the said suppression isneutralised by inducing expression of the DNA sequence(s) in said secondpolynucleotide sequence from said controllable promoter region byexternal application of an inducing substance thus making restoration ofsprouting of said tuber dependent on the application of the inducer, sothat sprouting in said transgenic potato is selectively induced orsuppressed.
 3. A method according to claim 1 wherein the DNA sequence(s)in said first polynucleotide sequence comprises a DNA sequence codingfor an inorganic pyrophosphatase derived from plant, bacterial or fugalsources, or an invertase derived from plant, bacterial or fungalsources.
 4. A method according to claim 1 wherein the DNA sequence(s) insaid second polynucleotide sequence comprises a DNA sequence which isselected from the group consisting of: a sense sequence with respect tosaid first DNA sequence, an anti-sense sequence with respect to saidfirst DNA sequence a partial sense sequence with respect to said firstDNA sequence and a DNA sequence which is capable of causing suppressionof said first DNA sequence.
 5. A method according to claim 1 wherein thetissue or organ selective promoter is the rolC promoter or a tuberpromoter.
 6. A method according to claim 1 wherein the DNA sequence(s)in the second polynucleotide sequence of the construct is under thecontrol of a controllable promoter region which may be inducedchemically by the application of an external chemical stimulus.
 7. Amethod according to claim 6 wherein the controllable promoter region isthe alcA/alcR or GST or ecdysone switch promoter.
 8. A method accordingto any one of claim 1 wherein said first polynucleotide sequencecomprises a further DNA sequence coding for an operator sequenceoperably linked to the first DNA sequence and the second polynucleotidesequence comprises a DNA sequence coding for a repressor protein capableof binding to said operator sequence.
 9. A method according to claim 8wherein said operator and repressor sequences comprise the lactose,tetracycline or lambda 434 operator/repressor sequences and mutantsthereof.